Neonatal Rat Liver Research Articles

Yinzhihuang Oral Liquid Ameliorates Hyperbilirubinemia Induced by δ-Aminolevulinic Acid and Novobiocin in Neonatal Rats.

Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti-hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ-Aminolevulinic acid (ALA, 200 mg/kg) or novobiocin (NOVO, 200 mg/kg). Oral treatment of YZH (80, 160 and 320 mg/kg) significantly decreased serum conjugated bilirubin levels in ALA-treated neonatal rats and serum unconjugated bilirubin levels in NOVO-treated neonatal rats, respectively. Additionally, pre-treatment of YZH also prevented the increase of serum bilirubin levels in both ALA- and NOVO-treated rats. Mechanistically, YZH significantly up-regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion-transporting polypeptide 1b2, Oatp1b2; multidrug resistance-associated protein 2, Mrp2) and bilirubin conjugation (UDP-glucuronosyltransferase 1a1, Ugt1a1). Additionally, YZH up-regulated the mRNA expression of cytochrome P450 1A1 (Cyp1a1), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4'-hydroxyacetophenone (4-HT), up-regulated the mRNA expression of AhR target genes (CYP1A1 and UGT1A1) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti-hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.

Effects of Experimentally Induced Nicotine on the Liver of Neonatal Albino Rat

Effects of Experimentally Induced Nicotine on the Liver of Neonatal Albino Rat

Effects of intravenous and oral di(2-ethylhexyl) phthalate (DEHP) and 20% Intralipid vehicle on neonatal rat testis, lung, liver, and kidney

The highest human exposures to the plasticizer di(2-ethylhexyl) phthalate (DEHP) occur through intravenous (iv) exposure from medical procedures. Rodent toxicity studies, mainly using oral exposures, have identified male reproductive toxicity after developmental exposure to DEHP as the primary concern. Other organs are also affected by DEHP and route may influence the degree of target organ involvement. Cammack et al. (2003) reported a critical study focused on testicular toxicity using oral and iv exposures of neonatal Sprague-Dawley rats to 60, 300, or 600 mg/kg body weight/day DEHP in Intralipid vehicle. The present study followed the same dosing paradigm and included assessment of additional organs to evaluate the potential utility of this design for DEHP alternatives. Reduction of testis weight was observed in all DEHP treatment groups and germ cell and Sertoli cell toxicity was observed at the two highest doses with both routes. Lung granulomas occurred in all iv DEHP groups, possibly related to increased fat particle size in DEHP lipid emulsions. Lung alveolar development was inhibited after both oral and iv high dose DEHP. Toxicity of oral Intralipid vehicle was observed in germ and Sertoli cells. The lack of such effects after iv vehicle exposure suggested that this may be a gut-mediated effect.

Hepatic Transcriptomic Patterns in the Neonatal Rat After Pentabromodiphenyl Ether Exposure.

Human exposure to pentabromodiphenyl ether (PBDE) mixture (DE-71) and its PBDE-47 congener can occur both in utero and during lactation. Here, we tested the hypothesis that PBDE-induced neonatal hepatic transcriptomic alterations in Wistar Han rat pups can inform on potential toxicity and carcinogenicity after longer term PBDE exposures. Wistar Han rat dams were exposed to either DE-71 or PBDE-47 daily from gestation day (GD 6) through postnatal day 4 (PND 4). Total plasma thyroxine (T4) was decreased in PND 4 pups. In liver, transcripts for CYPs and conjugation enzymes, Nrf2, and ABC transporters were upregulated. In general, the hepatic transcriptomic alterations after exposure to DE-71 or PBDE-47 were similar and provided early indicators of oxidative stress and metabolic alterations, key characteristics of toxicity processes. The transcriptional benchmark dose lower confidence limits of the most sensitive biological processes were lower for PBDE-47 than for the PBDE mixture. Neonatal rat liver transcriptomic data provide early indicators on molecular pathway alterations that may lead to toxicity and/or carcinogenicity if the exposures continue for longer durations. These early toxicogenomic indicators may be used to help prioritize chemicals for a more complete toxicity and cancer risk evaluation.

Evaluation of copper metabolism in neonatal rats by speciation analysis using liquid chromatography hyphenated to ICP mass spectrometry.

It is known that copper (Cu) is highly accumulated in several organs in the perinatal period, suggesting changes in Cu metabolism with development, although the precise mechanisms are still unclear. To elucidate the mechanisms underlying Cu accumulation in the organs of neonatal rats, we performed speciation analysis using high-performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry. In the neonatal rat liver immediately after birth, the Cu concentration was elevated 10-fold compared to that in the juvenile rat liver. Most of the accumulated Cu was bound to metallothionein, although Cu in Cu, zinc-superoxide dismutase (SOD) was reduced. Contrary to the hepatic Cu accumulation, the serum Cu concentrations in the neonatal rats were low due to the decreased amount of Cu bound to ceruloplasmin. The mRNA expression of antioxidant protein 1 (Atox1), a Cu chaperone that transports Cu to Atp7b, remained low up to two weeks after birth. These results suggest that Cu accumulation in the neonatal rat liver is caused by the low expression of Atox1, and the accumulation is useful to distribute Cu to Cu-containing anti-oxidative enzymes (e.g., SOD and Atox1) after respiration starts.

Brain Distribution and Metabolism of Flupirtine, a Nonopioid Analgesic Drug with Antiseizure Effects, in Neonatal Rats.

Flupirtine, a nonopioid analgesic drug, is effective in treating neonatal seizures. However, its brain delivery and pharmacokinetics are unknown in neonatal mammals. The purpose of this study was to determine the pharmacokinetics of flupirtine and the formation of its active metabolite D-13223 in various tissues such as brain in neonate animals. On postnatal day 7, rat pups received 25 mg/kg of flupirtine intraperitoneally. Liver; heart; kidney; lung; spleen; retina; serum; and brain regions hippocampus, cortex, and the remaining brain (devoid of cerebellum) were harvested up to 24-h postdosing. An LC-MS/MS assay was developed to quantify flupirtine and D-13223. Flupirtine was delivered to all tissues assessed, with the highest area under the concentration vs. time curve (AUC0–24h) in liver (488 µg·h/g tissue) and the lowest in spleen (82 µg·h/g tissue). Flupirtine reached the brain, including the hippocampus and cortex, within 1 h of dosing and persisted at 24 h. Flupirtine AUC in various brain regions was approximately 195 µg·h/g tissue. The half-life of flupirtine in various tissues ranged from 3.1 to 5.2 h. D-13223 was formed in vivo and detected in all tissues assessed, with the concentrations being the highest in the liver. Incubation of isolated neonatal rat liver, heart, kidney, lung, spleen, whole eye, serum, or whole brain with flupirtine for 3 h at 37 °C formed D-13223 in all tissues, except serum. D-13223 formation was the highest in isolated liver tissue. Tissue partition coefficients based on isolated tissue uptake correlated well with in vivo tissue:serum drug exposure ratios. Thus, flupirtine reaches the target brain tissues from the systemic route in neonatal rats, and brain tissue forms the active metabolite D-13223.

Intermittent hypoxia suppression of growth hormone and insulin-like growth factor-I in the neonatal rat liver

ObjectivesExtremely low gestational age neonates with chronic lung disease requiring oxygen therapy frequently experience fluctuations in arterial oxygen saturation or intermittent hypoxia (IH). These infants are at risk for multi-organ developmental delay, reduced growth, and short stature. The growth hormone (GH)/insulin-like growth factor-I (IGF-1) system, an important hormonal regulator of lipid and carbohydrate metabolism, promotes neonatal growth and development. We tested the hypothesis that increasing episodes of IH delay neonatal growth by influencing the GH/IGF-I axis. DesignNewborn rats were exposed to 2, 4, 6, 8, 10, or 12 hypoxic episodes (12% O2) during hyperoxia (50% O2) from P0-P7, P0-P14 (IH), or allowed to recover from P7-P21 or P14-P21 (IHR) in room air (RA). RA littermates at P7, P14, and P21 served as RA controls; and groups exposed to hyperoxia only (50% O2) served as zero IH controls. Histopathology of the liver; hepatic levels of GH, GHBP, IGF-I, IGFBP-3, and leptin; and immunoreactivities of GH, GHR, IGF-I and IGF-IR were determined. ResultsPathological findings of the liver, including cellular swelling, steatosis, necrosis and focal sinusoid congestion were seen in IH, and were particularly severe in the P7 animals. Hepatic GH levels were significantly suppressed in the IH groups exposed to 6–12 hypoxic episodes per day and were not normalized during IHR. Deficits in the GH levels were associated with reduced body length and increase body weight during IHR suggesting increased adiposity and catchup fat. Catchup fat was also associated with elevations in GHBP, IGF-I, leptin. ConclusionsIH significantly impairs hepatic GH/IGF-1 signaling during the first few weeks of life, which is likely responsible for hepatic GH resistance, increased body fat, and hepatic steatosis. These hormonal perturbations may contribute to long-term organ and body growth impairment, and metabolic dysfunction in preterm infants experiencing frequent IH and/or apneic episodes.

Liver Scaffolds Support Survival and Metabolic Function of Multilineage Neonatal Allogenic Cells.

Organ scaffold bioengineering is currently limited by the inability to effectively repopulate the scaffold with appropriately distributed functional cells. We examined the feasibility of a decellularized liver scaffold to support the growth and function of multilineage allogenic cells derived from either adult or neonatal liver cells. Cell slurries from neonatal and adult rat livers containing hepatocytes, cholangiocytes, and endothelial cells were introduced into decellularized adult rat liver scaffolds via the bile duct. Recellularized grafts were perfused with cell growth medium through the portal vein for 7 days. Concurrently, the same cell slurries were incubated on culture dishes. Albumin levels were measured from graft perfusates and cell culture media. Immunofluorescent assays were used to verify the colocalization of cholangiocytes, hepatocytes, endothelial cells, and Kupffer cells in the recellularized grafts by using anti-CK7, anti-hepatocyte antigen, anti-CD34, and anti-CD68, respectively. More robust albumin production was detected in the perfusate of scaffolds recellularized with a neonatal liver cell slurry compared with those with an adult liver cell slurry. The perfusates from all recellularized grafts showed increasing albumin concentration over 7 days; higher levels were detected in the constructs compared with the cell culture. Scaffolds seeded with a neonatal liver cell slurry showed the presence of hepatocytes, cholangiocytes, endothelial cells, and Kupffer cells. Results demonstrated the superiority of neonatal allogenic cells over adult cells of the same origin, possibly because of their pluripotent behavior. Liver bio-scaffolds supported the growth of four different liver cell lines. Recellularized grafts exhibited preserved functionality as demonstrated by albumin production, and constructs seeded with a neonatal cell slurry demonstrated proliferation on Ki-67 assay, thus representing a promising model for a transplantable construct.

Hepatic Uptake of Vitamin A is Independent of Retinoic Acid Pretreatment in Neonatal Rats

Previous studies have shown that total retinoid concentration in the liver of neonatal rats would increase in response to the VARA supplementation, which is a combination of vitamin A (VA) and retinoic acid (RA). Later studies have further demonstrated that a single dose of VA may stimulate the hepatic uptake, which in turn, significantly elevated the liver total retinol concentration. In current study, we gave the neonatal rats the RA treatment prior to the VA supplementation, and examined the distinct effect of RA on VA metabolism and kinetics in the liver tissue of these young animals. Sprague‐Dawley male and female rats were mated, and the dams were fed a VA‐marginal diet (0.35 mg retinol equivalents/kg diet) and were allowed to consume food and water ad libitum. On postnatal day (P) 2 and 3, pups from experimental group received an oral dose of RA (0.625μg/g body weight), while other pups received canola oil as the control treatment. Then, an oral dose of VA (6 μg retinyl palmitate/g body weight), containing 2 μCi of [3H]‐retinol as a tracer, was administered to all pups on P4. Pups (n = 4–6/treatment/time point) were euthanized from 25 min to 30 h, a total of 12 time points, after dose administration and the liver were collected. Tissue total retinoid mass was determined by ultra‐performance liquid chromatography (UPLC), and the tissue tracer level was measured by liquid scintillation counting. Results showed that in both oil‐pretreated and RA‐pretreated groups, the fraction of retinyl ester and retinol entering the liver increased continuously and peaked at 10 h. After that, the fraction of retinol slightly declined and remained at the same level until the end of the study. However, the fraction of retinyl ester decreased gradually until the 20hr time point and showed a trend of increase afterward. At the same time, liver vitamin A content of both groups increased gradually and peaked firstly at 10 h, after a slight decrease it reached a second peak at 20 h and declined in the following period of time. In conclusion, our findings indicate that liver uptake of dietary vitamin A may be tightly controlled and would not be affected by RA supplementation.Support or Funding InformationNIH grant HD006982

Chronic Intermittent Hypoxia Causes Lipid Peroxidation and Altered Phase 1 Drug Metabolizing Enzymes in the Neonatal Rat Liver

Critically ill preterm neonates requiring oxygen therapy often experience frequent apneas with intermittent hypoxia (IH). IH-induced oxidative stress causes lipid peroxidation, which targets the liver and contributes to toxic drug reactions. We tested the hypothesis that incremental IH episodes induce oxidative damage in the neonatal liver and alter the expression of genes that regulate drug metabolism. Newborn rats were exposed to increasing IH episodes (12% O2) during hyperoxia (50% O2), or placed in room air (RA) until postnatal day 21 (P21) for recovery from IH (IHR). RA littermates served as controls, and pups exposed to 50% O2 served as hyperoxia controls. Hepatic histopathology, biomarkers of oxidative stress and oxidative DNA damage, antioxidants, and expression of genes that regulate drug metabolism were assessed. Oxidative stress and DNA damage, evidenced by 8-isoprostaglandin F2α (8-isoPGF2α) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG), respectively, increased as a function of IH episodes, and was associated with decreased superoxide dismutase (SOD) and increased catalase activities. Pathological changes including cellular swelling, steatosis, necrosis, and focal sinusoid congestion were seen in IH, but not in IHR. Similarly, IH was associated with upregulation of several genes involved in DNA repair, which were downregulated during IHR. Of the genes involved in drug metabolism, aldehyde dehydrogenases (involved in lipid peroxidation) and cytochrome P450 (CYP) genes of the 2C family (involved in oxidative stress) were robustly upregulated both in IH and in IHR. Hepatic oxidative stress and lipid peroxidation occurring in response to chronic IH have implications for preterm infants, and may explain, in part, the pharmacokinetic variations and drug toxicities in this vulnerable population.

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

ABSTRACTRecent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.

A proteomic study on liver impairment in rat pups induced by maternal microcystin-LR exposure

There is mounting evidence indicating that microcystins (MCs) are heptapeptide toxins. Recent studies have also shown that MCLR can transfer from mother to offspring, but it is unclear whether maternal MCLR can influence the liver of offspring or not. In this study, pregnant SD rats were injected intraperitoneally with a saline solution (control) or 10 μg/kg MCLR per day from gestational day 8 (GD8) to postnatal day 15 (PD15) for a total of 4 weeks. 2-DE and MALDI-TOF-TOF mass spectrometry were used to screen for MCLR target proteins in the livers of rat pups. Our results demonstrated that MCLR could accumulate in the livers of neonatal rats. Proteomics studies also showed that MCLR significantly influenced many proteins, including those involved in the cytoskeleton, metabolism and particularly oxidative stress. In addition, MCLR induced cellular structural damage and resulted in the production of intracellular reactive oxygen species (ROS) and lipid peroxidation. Moreover, protein phosphatase (PP) activity was inhibited and some serum biochemistry parameters were altered. These results suggest an early molecular mechanism behind the hepatotoxicity induced by maternal MC exposure and highlight the importance of monitoring MC concentrations in new-born mammals.

Cerebral Area Differential Redox Response of Neonatal Rats to Selenite-Induced Oxidative Stress and to Concurrent Administration of Highbush Blueberry Leaf Polyphenols.

Our goal was to delineate the mechanisms of selenite-induced oxidative stress in neonatal rats and investigate the potential of blueberry leaf polyphenols to counteract the induced stress. Vaccinium corymbosum leaf decoction (BLD) was analyzed by UPLC-MS and LC-DAD, along with its in vitro antioxidant activity (DPPH radical scavenging, FRAP, ferrous chelation). Newborn suckling Wistar rats were randomly divided into three groups: 'Se' and 'SeBLD' received 20 μmol Na2SeO3/kg BW subcutaneously (PN day 10); 'SeBLD' received 100 mg dry BLD/kg BW intraperitoneally (PN11 and 12) and Group 'C' received normal saline. Βiochemical analysis revealed tissue-specific effects of selenite. Brain as a whole was more resistant to selenite toxicity in comparison to liver; midbrain and cerebellum were in general not affected, but cortex was moderately disturbed. Liver lipid peroxidation, GSH, SOD, CAT, GPx were significantly affected, whereas proteolytic activity was not. BLD, which is rich in chlorogenic acid and flavonols (especially quercetin derivatives), exerted significant antioxidant protective effects in all regions. In conclusion, we provide for the first time an insight to the neonatal rat cerebral and liver redox response against a toxic selenite dose and blueberry leaf polyphenols.

The role of ghrelin on apoptosis, cell proliferation and oxidant-antioxidant system in the liver of neonatal diabetic rats.

Ghrelin is a multifunctional peptide hormone which stimulates appetite and regulates glucose metabolism and adipogenesis. The purpose of this study was to investigate whether ghrelin has protective effects in the liver of streptozocin (STZ) diabetic rats or not. Wistar-type neonatal rats were divided into four groups: I. Controls, II. Ghrelin administrated controls, III. STZ-diabetic rats, and IV. Ghrelin administrated diabetic rats. On the second day after birth, 100 mg/kg STZ was administered intraperitoneally in a single dose to induce diabetes in rats. 100 µg/kg/day ghrelin was administrated to rats subcutaneously for 4 weeks. Ghrelin administration improved histopathologic changes in STZ-diabetic liver. Obestatin immunoreactivity has been shown in livers of neonatal rats. The immunoreactivity of obestatin increased in diabetic rats and a decline was observed in ghrelin administrated diabetic rats. Caspase 8 and 3 immunoreactivities increased in diabetic rats; however, ghrelin administration differently affected caspases 8 and 3 immunoreactivities. Proliferating cell nuclear antigen immunoreactivities decreased in diabetic rats and in ghrelin administrated diabetic rats. Serum alanine (P < 0.05) and aspartate transaminase (P < 0.0001) and serum alkaline phosphatase (P < 0.0001) activities were decreased in ghrelin administrated diabetic rats compared to the diabetic rats. Gamma glutamyl transferase activity (P < 0.001) decreased in ghrelin administrated diabetic rats compared to the diabetic rats. The response of antioxidants including glutathione levels, catalase and superoxide dismutase activities were altered in ghrelin administrated diabetic rats. Our findings indicate that ghrelin administration affects hepatic functions in neonatal diabetic rats and might be considered as a therapeutic agent.

Vitamin A supplementation promotes the uptake and tissue storage of vitamin A in a kinetic study of neonatal rats (645.21)

Previous studies conducted in our laboratory have shown that supplementing neonatal rats with a combination of vitamin A (VA) and retinoic acid increases total retinol (ROH) concentration in plasma, liver and lung. The objective of the present study was to examine the effect of VA supplementation, in the absence of retinoic acid, on the metabolism and kinetics of total ROH in plasma and liver of neonatal rats. On postnatal d 4, pups (n = 103) received an oral dose of either VA (6 μg of retinyl palmitate/g of body weight) or canola oil, both containing 2.5 μCi of [3H]retinol acting as the tracer. Subsequently, pups (n = 4/group/time) were euthanized at 13 time‐points from 30 min to 24 d after dose administration. VA supplementation transiently increased total ROH concentration in plasma, due mainly to an elevation of retinyl esters, which disappeared 15 h after dosing. In the liver, total ROH concentration remained significantly elevated in the VA group until d 18 after dosing. VA also increased the amount of [3H]retinol present in the liver from d 8 to d 12 and enhanced the clearance of [3H]retinol from plasma on d 1 following dose administration. The results indicate that VA supplementation may stimulate the uptake of VA by the liver for storage during the neonatal period.Grant Funding Source: NIH HD‐066982

Maternal high-fat diet programs Wnt genes through histone modification in the liver of neonatal rats

Maternal high-fat (HF) diets during gestation and lactation have been shown to contribute to metabolic disorders in offspring. Molecular and epigenetic mechanisms underlying this connection may be essential for the prevention and treatment of the fetal origins of metabolic diseases. The current study examined the impact of maternal HF diets on Wnt signaling and histone modification in offspring. Time-pregnant Sprague-Dawley rats were fed either control diet or HF diet during gestation and lactation and then the neonatal offspring of both groups were investigated. The neonatal offspring born to dams fed on HF diets exhibited increases in serum glucose and liver triglyceride levels. Maternal exposure to the HF diet also repressed the mRNA expression of Wnt1 and nuclear β-catenin protein in the liver of offspring. The altered Wnt1 gene expression may be due to the changes of acetylation of H4 at its promoter as well as acetylation of H4 and methylation of H3K9 at coding region. Maternal exposure to the HF diet induced suppression of the Wnt/β-catenin signaling pathway through histone modification, potentially increasing the risk of metabolic syndrome.

Metabolic development in the liver and the implications of the n-3 fatty acid supply

The n-3 fatty acids contribute to regulation of hepatic fatty acid oxidation and synthesis in adults and accumulate in fetal and infant liver in variable amounts depending on the maternal diet fat composition. Using 2D gel proteomics and matrix-assisted laser desorption/ionization time of flight mass spectrometry, we recently identified altered abundance of proteins associated with glucose and amino acid metabolism in neonatal rat liver with increased n-3 fatty acids. Here, we extend studies on n-3 fatty acids in hepatic metabolic development to targeted gene and metabolite analyses and map the results into metabolic pathways to consider the role of n-3 fatty acids in glucose, fatty acid, and amino metabolism. Feeding rats 1.5% compared with <0.1% energy 18:3n-3 during gestation led to higher 20:5n-3 and 22:6n-3 in 3-day-old offspring liver, higher serine hydroxymethyltransferase, carnitine palmitoyl transferase, and acyl CoA oxidase and lower pyruvate kinase and stearoyl CoA desaturase gene expression, with higher cholesterol, NADPH and glutathione, and lower glycine (P < 0.05). Integration of the results suggests that the n-3 fatty acids may be important in facilitating hepatic metabolic adaptation from in utero nutrition to the postnatal high-fat milk diet, by increasing fatty acid oxidation and directing glucose and amino acids to anabolic pathways.

Administration of capsule-selective endosialidase E minimizes upregulation of organ gene expression induced by experimental systemic infection with Escherichia coli K1

Many neurotropic strains of Escherichia coli cause potentially lethal bacteraemia and meningitis in newborn infants by virtue of their capacity to elaborate the protective polysialic acid (polySia) K1 capsule. Recombinant capsule depolymerase, endosialidase E (endoE), selectively removes polySia from the bacterial surface; when administered intraperitoneally to infected neonatal rats, the enzyme interrupts the transit of E. coli K1 from gut to brain via the blood circulation and prevents death from systemic infection. We now show that experimental E. coli K1 infection is accompanied by extensive modulation of host gene expression in the liver, spleen and brain tissues of neonatal rats. Bacterial invasion of the brain resulted in a threefold or greater upregulation of approximately 400 genes, a large number of which were associated with the induction of inflammation and the immune and stress responses: these included genes encoding C-X-C and C-C chemokines, lipocalins, cytokines, apolipoproteins and enzymes involved in the synthesis of low-molecular-mass inflammatory mediators. Administration of a single dose of endoE, 24 h after initiation of systemic infection, markedly reduced, but did not completely abrogate, these changes in gene expression, suggesting that attenuation of E. coli K1 virulence by removal of the polySia capsule may minimize the attendant inflammatory processes that contribute to poor outcome in these severe systemic infections.

Identification of novel protein targets regulated by maternal dietary fatty acid composition in neonatal rat liver

Polyunsaturated fatty acids regulate metabolic pathways, which in early development could have important consequences to adaptation to extra-uterine life and programming of metabolic pathways. Female rats were fed one of two diets identical in all nutrients, except that the fat in one diet was high in unsaturated fatty acids (UFA) and the other low UFA, through gestation and lactation. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis of protein extracts from 3-day old pup liver resolved over 800 proteins. Employing MALDI-TOF MS and peptide mapping we identified 11 proteins that differed more than three-fold between the groups, 10 up regulated and one down regulated in the high UFA group. The up-regulated proteins included fructose-1,6-bisphosphatase 1, glycerol-3-phosphate dehydrogenase, galactokinase 1, 40S ribosomal protein SA, elongation factor 1-gamma, protein disulfide-isomerase A6, catalase, cytokeratin-8 and 60 kDa heat shock protein, and the down-regulated protein was argininosuccinate synthase, none having been previously reported to be regulated by fatty acids in the developing liver. We further determined that fructose-1,6-biphosphatase is acetylated at the N-terminus. We demonstrate that early fatty acid nutrition impacts hepatic metabolic pathways relevant to gluconeogenesis, redox balance and nitric oxide signaling.

Targeted suppression of heme oxygenase-1 by small interference RNAs inhibits the production of bilirubin in neonatal rat with hyperbilirubinemia

BackgroundExcessive accumulation of bilirubin contributes to neonatal hyperbilirubinemia in rats. Heme oxygenase (HO) is one of the rate-limiting enzymes in catabolizing heme to bilirubin. In the present study, we investigated whether suppression of rat HO-1 (rHO-1) expression by small interference RNAs (siRNAs) reduces bilirubin levels in hyperbilirubinemic rats.ResultsFour pairs of siRNA targeting rHO-1 mRNA were introduced into BRL cells and compared for their inhibitory effect on the expression of rHO-1 gene and production of rHO-1 protein. The siRNA exhibiting the most potent effect on HO-1 expression and activity was then administered intraperitoneally to 7 to 9-day-old rats with hyperbilirubinemia. The siRNA distributed mostly in the liver and spleen of neonatal rat. Serum bilirubin levels and hepatic HO-1 expression were further evaluated. Systemic treatment of siRNA targeting rHO-1 reduced hepatic HO-1 expression and decreased the serum bilirubin levels in a time- and dose-dependent manner, and siRNA decreased the indirect bilirubin levels more effectively than Sn-protoporphyrin (SnPP), an HO-1 inhibitor.ConclusionsiRNA targeting rHO-l attenuates hepatic HO-1 expression and serum bilirubin levels. Thus this study provides a novel therapeutic rationale for the prevention and treatment of neonatal hyperbilirubinemia.