Novel preparation of bilirubin-encapsulated pluronic F-127 nanoparticles as a potential biomaterial for wound healing.

Cutaneous wounds deteriorate the health of patients and liable for high economic loss. Previous studies showed promising wound healing potentials of bilirubin, however, this macromolecule constrained with poor water solubility and skin penetration. In this study, Pluronic F-127, a non-ionic copolymer surfactant, was used for the encapsulation of the wound healing agent the bilirubin. With this strategy, spherical shaped bilirubin nanoparticles of ∼100-150 nm with zeta potential ranging from -13.43 ± 0.56 to -17.53 ± 0.43 mV were obtained. Topical applications of bilirubin nanoparticle (0.3%) on cutaneous wounds of rats showed promising wound healing in comparison with other topical treatments. This topical nano-formulation also modulates the cytokine and growth factor responses in the treated group. On day 7 of healing, bilirubin nanoparticles treatment significantly reduced TNF-α and increased IL-10 levels with increased VEGF and TGF-ß1 expressions. Simultaneously, prominent pro-healing activities could be observed histopathologically. These include increased blood vessels, reduced inflammatory cells, more myofibroblasts, increased deposition of collagen fibres, and early re-epithelialization. The changes were prominent in bilirubin nanoparticles (0.3%) treated group indicating better granulation tissue, quality of healing and wound maturity. In conclusion, the proposed new encapsulated bilirubin nanoparticles strategy significantly improved wound healing by modulation of cytokines and growth factors response in comparison with native bulk bilirubin. These observations support its potential as a novel biomaterial for wound healing in the future.

Folate-Gold-Bilirubin Nanoconjugate Induces Apoptotic Death in Multidrug-Resistant Oral Carcinoma Cells.

BACKGROUND: Gold nanoparticles (GNPs) are receiving increasing attention as drug delivery carriers due to their high surface-to-volume ratio, hydrophilicity, and functionality. Drug delivery by nanocarriers has the potential to bypass P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) by altering the drug internalization mechanism and/or intracellular release pattern, inhibiting the activity of ABC-transporter efflux pumps, or downregulating the expression of genes responsible for the activity of efflux pumps. OBJECTIVE: We developed a folate-gold-bilirubin (FGB) nanoconjugate to reverse MDR in P-expressing KB-ChR-8-5 cells. METHODS: The P-gp overexpressing KB-ChR-8-5 cells were incubated with the FGB nanoconjugate, bilirubin, or GNPs. Various cellular endpoints, such as cytotoxicity, ROS generation, DNA damage, and apoptosis, were analyzed using analytical methods. Further, a KB-ChR-8-5 cell-bearing tumor xenograft was developed and the anticancer potential of the prepared FGB nanoparticles was compared to that of bilirubin or GNPs in this preclinical model. RESULTS: The FGB nanoconjugate was found to be a stronger inhibitor of the viability of multidrug-resistant KB-ChR-8-5 cells than bilirubin and GNPs treatment alone. The nanoconjugate induced reactive oxygen species (ROS) formation, DNA strand breaks, and apoptotic morphological changes in the P-gp-overexpressing drug-resistant cells to a greater degree than bilirubin treatment alone. Also, the FGB nanoparticles led to stronger suppression of tumor development in the KB-ChR-8-5 xenograft mouse model than achieved with bilirubin treatment alone. Thus, the present results suggest that the FGB nanoconjugate suppresses tumor growth in drug-resistant tumor cells by inducing apoptotic cell death. CONCLUSION: FGB nanoparticles significantly inhibit tumor growth, probably through the folate receptor, which is highly expressed in KB cells. Hence, folate-gold-bilirubin nanoparticles could be a promising agent for inducing apoptosis in P-gp-overexpressing drug-resistant cancer cells.

Unconjugated bilirubin elevation impairs the function and expression of breast cancer resistance protein (BCRP) at the blood-brain barrier in bile duct-ligated rats.

AIM: Liver failure is associated with dyshomeostasis of efflux transporters at the blood-brain barrier (BBB), which contributes to hepatic encephalopathy. In this study we examined whether breast cancer resistance protein (BCRP), a major efflux transporter at the BBB, was altered during liver failure in rats. METHODS: Rats underwent bile duct ligation (BDL) surgery, and then were sacrificed after intravenous injection of prazosin on d3, d7 and d14. The brains and blood samples were collected. BCRP function at the BBB was assessed by the brain-to-plasma prazosin concentration ratio; Evans Blue extravasation in the brain tissues was used as an indicator of BBB integrity. The protein levels of BCRP in the brain tissues were detected. Human cerebral microvessel endothelial cells (HCMEC/D3) and Madin-Darby canine kidney cells expressing human BCRP (MDCK-BCRP) were tested in vitro. In addition, hyperbilirubinemia (HB) was induced in rats by intravenous injection of unconjugated bilirubin (UCB). RESULTS: BDL rats exhibited progressive decline of liver function and HB from d3 to d14. In the brain tissues of BDL rats, both the function and protein levels of BCRP were progressively decreased, whereas the BBB integrity was intact. Furthermore, BDL rat serum significantly decreased BCRP function and protein levels in HCMEC/D3 cells. Among the abnormally altered components in BDL rat serum tested, UCB (10, 25 µmol/L) dose-dependently inhibit BCRP function and protein levels in HCMEC/D3 cells, whereas 3 bile acids (CDCA, UDCA and DCA) had no effect. Similar results were obtained in MDCK-BCRP cells and in the brains of HB rats. Correlation analysis revealed that UCB levels were negatively correlated with BCRP expression in the brain tissues of BDL rats and HB rats as well as in two types of cells tested in vitro. CONCLUSION: UCB elevation in BDL rats impairs the function and expression of BCRP at the BBB, thus contributing to hepatic encephalopathy.

Bilirubin modulated cytokines, growth factors and angiogenesis to improve cutaneous wound healing process in diabetic rats.

Bilirubin has shown cutaneous wound healing potential in some preliminary studies. Here we hypothesize that bilirubin facilitates wound healing in diabetic rats by modulating important healing factors/candidates and antioxidant parameters in a time-dependent manner. Diabetes was induced in male Wistar rats by streptozotocin. In all diabetic rats wounds were created under pentobarbitone anesthesia. All the rats were divided into two groups, of which one (control) was treated with ointment base and other with bilirubin ointment (0.3%). Wound closer measurement and tissue collection were done on days 3, 7, 14 and 19 post-wounding. The relative expressions of hypoxia inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 alpha (SDF-1α), transforming growth factor- beta1 (TGF-ß1()), tumor necrosis factor-α (TNF-α) and interlukin-10 (IL-10) mRNA and proteins and the mRNA of interlukin-1 beta (IL-1ß) and matrix metalloprteinase-9 (MMP-9) were determined in the wound tissues. CD-31 staining and collagen content were evaluated by immunohistochemistry and picrosirius red staining, respectively. Histopathological changes were assessed by H&E staining. The per cent wound closer was significantly higher from day 7 onwards in bilirubin-treated rats. HIF-1α, VEGF, SDF-1α, TGF-ß1, IL-10 mRNA and protein levels were significantly higher on days 3, 7 and 14 in bilirubin-treated rats. The mRNA expression and protein level of TNF-α and the mRNA of IL-1ß and MMP-9 were progressively and markedly reduced in bilirubin-treated rats. The collagen deposition and formation of blood vessels were greater in bilirubin-treated rats. Bilirubin markedly facilitated cutaneous wound healing in diabetic rats by modulating growth factors, cytokines, neovasculogenesis and collagen contents to the wound site. Topical application of bilirubin ointment might be of great use in cutaneous wound healing in diabetic patients.

Proliferative activity of liver growth factor is associated with an improvement of cigarette smoke-induced emphysema in mice.

Cigarette smoke (CS)-induced emphysema is a major component of chronic obstructive pulmonary disease (COPD). COPD treatment is based on the administration of bronchodilators and corticosteroids to control symptoms and exacerbations, however, to date, there are no effective therapies to reverse disease progression. Liver growth factor (LGF) is an albumin-bilirubin complex with mitogenic properties, whose therapeutic effects have previously been reported in a model of emphysema and several rodent models of human disease. To approach the therapeutic effect of LGF in a model of previously established emphysema, morphometric and lung function parameters, matrix metalloproteinase (MMP) activity and the expression of several markers, such as VEGF, PCNA, 3NT and Nrf2, were assessed in air-exposed and CS-exposed C57BL/6J male mice with and without intraperitoneal (i.p.) injection of LGF. CS-exposed mice presented a significant enlargement of alveolar spaces, higher alveolar internal area and loss of lung function that correlated with higher MMP activity, higher expression of 3NT and lower expression of VEGF. CS-exposed mice injected with LGF, showed an amelioration of emphysema and improved lung function, which correlated with lower MMP activity and 3NT expression and higher levels of VEGF, PCNA and Nrf2. Taken together, this study suggests that LGF administration ameliorates CS-induced emphysema, highlights the ability of LGF to promote alveolar cell proliferation and may be a promising strategy to revert COPD progression.

Bilirubin attenuates the renal tubular injury by inhibition of oxidative stress and apoptosis.

BACKGROUND: Bilirubin (BIL) has been recognized as an endogenous antioxidant that shows a protective effect for cardiorenal diseases. We investigated whether administration of BIL had a protective effect on cyclosporine (CsA)-induced nephropathy (CIN), and examined the effects of BIL on the oxidative stress and apoptosis. METHODS: BIL was pretreated intraperitoneally three times for a week (60 mg/kg), and CsA was injected for 4 weeks (15 mg/kg/day, subcutaneous). Proximal tubular epithelial (HK2) cells were pretreated with 0.1mg/ml of BIL for 24 hours, and then treated with 20 µM of CsA for another 24 hours. RESULTS: CsA induced marked increases in urine kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL) concentrations (P < 0.05). BIL reduced urine Kim-1 in CIN (P < 0.05), while urine NGAL exhibited a decreasing tendency. In CsA-treated rat kidneys, the protein expression of NOX4 and p22phox was reduced by BIL (P < 0.05). BIL ameliorated CsA-induced arteriolopathy, tubulointerstitial fibrosis, tubular injury, and the apoptosis examined by TUNEL assay (P < 0.01). In HK2 cells, BIL reduced intracellular reactive oxygen species in CsA-treated cells. CsA increased the protein expression of bax, cleaved caspase-9, caspase-3 and the activity of caspase-3; however, the anti-apoptotic bcl-2 protein was reduced. These changes were recovered by BIL (P < 0.05). CONCLUSIONS: The direct administration of BIL protected against CsA-induced tubular injury via inhibition of oxidative stress and apoptosis.

Application of quantitative time-lapse imaging (QTLI) for evaluation of Mrp2-based drug-drug interaction induced by liver metabolites.

We previously reported a quantitative time-lapse imaging (QTLI)-based analysis method to assess drug-drug interactions (DDI) at multidrug resistance-associated protein 2 (Mrp2) in rat sandwich-cultured hepatocyte (SCH) system, utilizing the fluorescent Mrp2 substrate, 5-(and 6)-carboxy-2',7'-dichlorofluorescein (CDF). Here, we aimed to examine the feasibility of using QTLI to evaluate DDI involving drug metabolite(s) generated in hepatocytes. We used estradiol (E2) and bilirubin as model compounds; both are not substrates of MRP2, whereas their hepatic metabolites, estradiol-17ß-glucuronide (E17G) or bilirubin glucuronides, are known to be its substrates as well as inhibitors. When rat SCHs were pre-exposed with E2, fluorescence of CDF accumulated in bile canaliculi decreased depending upon both the duration of pre-exposure and the concentration of extracellular E2. The decrease corresponded with the increase in intracellular concentration of E17G in hepatocytes. Furthermore, cytotoxicity of vinblastine, a substrate of MRP2, was enhanced in SCHs treated with E2. Similarly, CDF accumulated in bile canaliculi was significantly reduced in rat SCHs pre-exposed with bilirubin. In conclusion, these results suggest that phase II biotransformation of a competitor is reflected in alteration of MRP2-mediated CDF transport detected in QTLI. The QTLI might provide a convenient platform to evaluate transporter-based DDIs involving hepatic metabolites of drug candidates without the need to identify the metabolites.

Intracellular accumulation of bilirubin as a defense mechanism against increased oxidative stress.

Antioxidant, anti-inflammatory and anti-atherogenic effects have been associated with elevations of unconjugated bilirubin (UCB) in serum and with the induction of heme oxygenase-1 (HO-1), the rate-limiting enzyme in UCB synthesis. The aim of this study was to investigate the intracellular metabolism and antioxidant properties of UCB in human hepatoblastoma HepG2 cells and tissues of Wistar rats exposed to oxidative stressors and lipopolysaccharide (LPS), respectively. Intracellular UCB concentrations in HepG2 cells correlated with its levels in culture media (p < 0.001) and diminished lipid peroxidation in a dose-dependent manner (p < 0.001). Moreover, induction of HO-1 with sodium arsenite led to 2.4-fold (p = 0.01) accumulation of intracellular UCB over basal level while sodium azide-derived oxidative stress resulted in a 60% drop (p < 0.001). This decrease was ameliorated by UCB elevation in media or by simultaneous induction of HO-1. In addition, hyperbilirubinemia and liver HO-1 induction in LPS-treated rats resulted in a 2-fold accumulation of tissue UCB (p = 0.01) associated with enhanced protection against lipid peroxidation (p = 0.02). In conclusion, hyperbilirubinemia and HO-1 induction associated with inflammation and oxidative stress increase intracellular concentrations of UCB, thus enhancing the protection of cellular lipids against peroxidation. Therefore, the previously reported protective effects of hyperbilirubinemia and HO-1 induction are at least in part due to intracellular accumulation of UCB.

Liver growth factor promotes the survival of grafted neural stem cells in a rat model of Parkinson's disease.

Neural stem cells (NSCs) with self-renewal and multilineage potential are considered good candidates for cell replacement of damaged nerve tissue. Several studies have focused on the ability of the neurotrophic factors coadministration to improve the efficiency of grafted NSCs. Liver growth factor (LGF) is an hepatic mitogen that promotes regeneration of damaged tissues, including brain tissue. It has neurogenic activity and has partially restored the nigrostriatal dopaminergic system in an experimental model of Parkinson's disease. Present results demonstrate that in the dopamine- depleted striatum of 6-hydroxydopamine-lesioned rats, grafted NSCs retained their ability to differentiate into neurons, astrocytes, and oligodendrocytes. NSCs also differentiated into microglia/macrophages and endothelial cells. Thus, 23 ± 5.6% of them were inmunoreactive for isolectin IB4, and a small population integrated into blood vessels, showing an endothelial-like morphology. Intrastriatal infusion of LGF promoted the viability of the implants, and favored their differentiation to an endothelial-like phenotype. Moreover, LGF infusion raised the expression of the anti-apoptotic protein Bcl-2 by 3.9 ± 0.9 fold without affecting the levels of the pro-apoptotic protein Bax. Since LGF-treated rats also showed a significant reduction in apomorphine-induced rotational behavior, our results suggest that administration of this factor might be a convenient treatment for Parkinson's disease cell replacement therapies based on NSCs transplantation.

Estudo comparativo entre a medida plasmática e transcutânea de bilirrubina em recém-nascidos / Comparative study between plasma and transcutaneous bilirubin measurements in newborns

OBJETIVO: Comparar as medidas transcutânea e plasmática da bilirrubina antes e durante a fototerapia, em área de pele exposta e coberta, analisando-se a associação com variáveis do recém-nascido (RN). MÉTODOS: Estudo de corte transversal, que avaliou 44 RN entre abril e outubro de 2008. Realizaram-se dosagens transcutâneas (região frontal e esternal) e plasmáticas da bilirrubina antes e 24 horas após o início da fototerapia. Tanto na região frontal como na esternal, ocluiu-se pequena região de pele e obteve-se a medida transcutânea da área coberta e de área adjacente exposta. Calculou-se a associação entre as medidas e variáveis do RN (peso, sexo, raça/cor, idade gestacional e pós-natal) e presença de fatores de risco para hiperbilirrubinemia significativa. RESULTADOS: Houve forte correlação entre a bilirrubina plasmática e a transcutânea, no momento da indicação e após 24 horas de fototerapia, nas regiões frontal e esternal, com intervalos de confiança estreitos tanto a 95 como a 99 por cento. Observou-se que, com relação à medida transcutânea na área coberta, 24 horas após o início da fototerapia, a medida esternal apresentou correlação mais forte com a plasmática (r=0,86; p<0,001). As variáveis do RN analisadas não interferiram nas medidas de bilirrubina. CONCLUSÕES: As dosagens transcutânea e plasmática apresentam correlação forte antes da fototerapia nas regiões frontal e esternal. Após 24 horas de fototerapia, a medida transcutânea esternal em área coberta apresentou melhor correlação.

OBJECTIVE: To compare transcutaneous and plasma bilirubin measurements before and during phototerapy, on exposed and covered body areas, and to verify the association of the obtained levels with neonatal characteristics. METHODS: This cross-sectional study enrolled 44 newborn infants from April to October 2008. Simultaneous plasmatic and transcutaneous (frontal and sternal regions) bilirubin assays were performed before and 24 hours after the beginning of phototerapy. On frontal and sternal regions, a small cover was placed and transcutaneous measurement was obtained from covered and exposed adjacent areas. The association between the measurements and neonatal weigh, sex, race, gestational and postnatal ages and risk factors for severe hyperbilirubinemia was calculated. RESULTS: There was a strong correlation between plasma and transcutaneous bilirubin assays measured in the frontal and sternal regions before the phototerapy, with narrow 95 and 99 percent confidence intervals. The covered sternal area presented the strongest correlation index 24 hours after phototherapy (r=0.86; p<0.001). No neonatal characteristic was significantly associated to the bilirubin levels. CONCLUSIONS: Transcutaneous measurements of frontal and sternal areas closely correlate with plasma bilirubin levels before starting phototerapy. After 24 hours on phototerapy, the transcutaneous sternal measurement on the covered area showed better correlation.

OBJETIVO: Comparar las medidas transcutánea y plasmática de la bilirrubina antes y durante la fototerapia, en área de piel expuesta y cubierta, analizando la asociación con variables del recién nacido (RN). MÉTODO: Estudio de corte transversal que analizó a 44 RN en el periodo de abril a octubre de 2008. Se realizaron dosificaciones transcutáneas (región frontal y esternal) y plasmática de bilirrubina antes y 24 horas después del inicio de la fototerapia. Tanto en la región frontal como en la esternal se ocluyó la pequeña región de piel y se obtuvo la medida transcutánea del área cubierta y del área adyacente expuesta. Se calculó la asociación entre las medidas y variables del RN (peso, sexo, raza/color, edad gestacional y postnatal y presencia de factores de riesgo para hiperbilirrubinemia significativa). RESULTADOS: La asociación entre bilirrubina plasmática y transcutánea, en el momento de la indicación y después de 24 horas de fototerapia en las regiones frontal y esternal fue muy homogénea, debido a la fuerte correlación y los intervalos de confianza estrechos, tanto a 95 por ciento como a 99 por ciento. Se observó, además, respecto a la medida transcutánea en el área cubierta, 24 horas después del inicio de la fototerapia, la medida en el área esternal presentó correlación más fuerte con la plasmática (r=0,8599; p=0,0001). Las variables del RN analizadas no interfirieron significativamente en las medidas de bilirrubina. CONCLUSIÓN: Las dosificaciones transcutánea y plasmática presentan correlación fuerte antes de la fototerapia en las regiones frontal y esternal. Tras 24 horas de la fototerapia, la medida transcutánea esternal en área cubierta presentó mejor correlación.

Hyperbilirubinemia reduces the streptozotocin-induced pancreatic damage through attenuating the oxidative stress in the Gunn rat.

Oxidative stress is an important pathogenic factor in diabetes. Bilirubin may serve a cytoprotective function as an anti-oxidant. The Gunn rat lacks the enzyme uridine-diphosphate glucuronosyltransferase that is responsible for conjugation of bilirubin, exhibiting elevation of plasma bilirubin. We examined the effect of hyperbilirubinemia on the pancreatic damage caused by streptozotocin (STZ) in the Gunn rat. Male Wistar rats and male Gunn rats were treated with STZ (WS and GS groups, respectively) or vehicle (WC and GC groups, respectively). All 5 rats in the WS group developed diabetes, defined as fasting blood glucose 300 mg/dL or more, at 3 days, whereas only 2 of the 5 GS rats became diabetic at 7 days after STZ injection. Without insulin supplement at 7 days after STZ injection, the WS group displayed higher levels of fasting blood glucose (510.3 ± 50.3 vs. 236.4 ± 42.5 mg/dL, p = 0.003) and HbA1c (5.0 ± 0.1 vs. 3.9 ± 0.1, p = 0.001), compared to those of GS group. In Wistar rats, STZ induced apoptosis of the pancreatic islet cells, accompanied with activation of NADPH oxidase and increased production of reactive oxygen species and nitric oxide, but not in Gunn rats. Moreover, in a rat insulinoma cell line (RIN-m5F), pre-treatment with bilirubin (0.1 mg/dL) decreased cell death and apoptosis caused by STZ, and also reduced H2O2 production. Considering the protective effect of hyperbilirubinemia against STZ-induced injury, we postulate that bilirubin could be a potential therapeutic modality for oxidative stress of pancreas islets.

Bilirubin protects grafts against nonspecific inflammation-induced injury in syngeneic intraportal islet transplantation.

Nonspecific inflammatory response is the major cause for failure of islet grafts at the early phase of intraportal islet transplantation (IPIT). Bilirubin, a natural product of heme catabolism, has displayed anti-oxidative and anti-inflammatory activities. The present study has demonstrated that bilirubin protected islet grafts by inhibiting nonspecific inflammatory response in a syngeneic rat model of IPIT. The inflammation-induced cell injury was mimicked by exposing cultured rat insulinoma INS-1 cells to cytokines (IL-1ß, TNF-α and IFN-γ) in in vitro assays. At appropriate lower concentrations, bilirubin significantly attenuated the reduced cell viability and enhanced cell apoptosis induced by cytokines, and protected the insulin secretory function of INS-1 cells. Diabetic inbred male Lewis rats induced by streptozotocin underwent IPIT at different islet equivalents (IEQs) (optimal dose of 1000, and suboptimal doses of 750 or 500), and bilirubin was administered to the recipients every 12 h, starting from one day before transplantation until 5 days after transplantation. Administration of bilirubin improved glucose control and enhanced glucose tolerance in diabetic recipients, and reduced the serum levels of inflammatory mediators including IL-1ß, TNF-α, soluble intercellular adhesion molecule 1, monocyte chemoattractant protein-1 and NO, and inhibited the infiltration of Kupffer cells into the islet grafts, and restored insulin-producing ability of transplanted islets.

Sodium taurocholate inhibits intestinal adenoma formation in APCMin/+ mice, potentially through activation of the farnesoid X receptor.

In light of clinical and biological evidence that bile constituents exert preventive effects against colorectal cancer, we evaluated the influence of oral bilirubin and sodium taurocholate (NaTC) on intestinal tumor formation in APC(Min/+) mice. Mice received bilirubin and/or bovine serum albumin (BSA) and NaTC in the drinking water for 8 weeks, after which the number, size and location of intestinal adenomas were determined. Tissue specimens were analyzed by light microscopy, TUNEL staining, immunohistochemistry for beta-catenin and Ki-67 and quantitative polymerase chain reaction for farnesoid X receptor (FXR)-dependent gene expression. Colon tumor formation also was assessed in azoxymethane (AOM)-treated hyperbilirubinemic Gunn (j/j) and wild-type (+/+) rats. Compared with untreated APC(Min/+) mice, the mean number of intestinal adenomas was markedly lower in both bilirubin (10.5 +/- 0.9 versus 37.0 +/- 5.2; +/-SEM; P < 0.001) and NaTC plus BSA (14.3 +/- 5.4; P = 0.01)-treated animals. Both treatment groups exhibited reduced levels of cellular proliferation in the ileum (by Ki-67 staining), but no differences in TUNEL staining or the percentage of beta-catenin-positive crypts. Bilirubin feeding reduced intestinal inducible nitric oxide synthase expression, but did not alter adenoma multiplicity in APC(Min/+) mice or in AOM-treated j/j versus +/+ rats. Mice receiving NaTC manifested increased intestinal expression of the FXR-regulated genes, Shp, FGF15 and IBABP, and a concomitant decrease in cyclin D1 message. Administering NaTC to APC(Min/+) mice causes a marked reduction in intestinal adenomas. We postulate that this effect is mediated through activation of FXR, leading to increased Shp expression and consequent downregulation of cyclin D1.

Mobilization of neural stem cells and generation of new neurons in 6-OHDA-lesioned rats by intracerebroventricular infusion of liver growth factor.

Neural stem cells with self-renewal and multilineage potential persist in the subventricular zone of the adult mammalian forebrain. These cells remain relatively quiescent but, under certain conditions, can be stimulated, giving rise to new neurons. Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and is useful for neuroregenerative therapies. The aim of this study was to investigate the potential neurogenic activity of LGF in the 6-hydroxydopamine rat model of Parkinson's disease. Proliferation was significantly increased in the subventricular zone and denervated striatum of rats receiving ICV LGF infusions, and 25% of the proliferating cells were doublecortin-positive neurons. Doublecortin-positive cells with the morphology of migrating neuroblasts were also observed in the dorsal and ventral regions of the striatum of LGF-infused animals. Moreover, some newly generated cells were neuronal nuclei-positive mature neurons. LGF also stimulated microglia and induced astrogliosis, both phenomena associated with generation and migration of new neurons in the adult brain. In summary, our study shows that LGF stimulates neurogenesis when applied intraventricularly in 6-hydroxydopamine-lesioned rats. Considering that this factor also promotes neuronal migration into damaged tissue, we propose LGF as a novel factor useful for neuronal replacement in neurodegenerative diseases.

Induction of human UGT1A1 by bilirubin through AhR dependent pathway.

UDP-glucuronosyltransferase1A1 (UGT1A1) plays a key role to conjugate bilirubin and preventing jaundice, but there is no report showing the induction of human UGT1A1 (UGT1A1) by bilirubin. In this report, we show findings of the induction of the reporter gene (-3475/+14) of UGT1A1 in HepG2 cells by bilirubin at 50 microM, 100 microM, with human aryl hydrocarbon receptor (hAhR). We confirmed that induction of the reporter gene by bilirubin is dependent on the position of the xenobiotic responsive element (XRE) (-3328/-3319) of UGT1A1, because the XRE deletion UGT1A1 gene did not respond to stimulation by a complex of bilirubin and hAhR. alpha-Naphthoflavone (alpha-NF) of a typical AhR antagonist at 50 microM inhibited induction by bilirubin, suggesting that bilirubin stimulates through binding with hAhR. Meanwhile, bilirubin itself did not stimulate the induction of AhR, because we detected no-elevation of the mRNA level of AhR by RT-PCR. These results indicate that the induction of UGT1A1 by bilirubin-AhR did not depend on the elevation of AhR but on ligand binding. From this result, we considered that high bilirubin in neonates must induce the elevation of UGT1A1 after birth to prevent jaundice, and bilirubin in adults also regulates the level of UGT1A1. This is the first report showing direct induction of UGT1A1 by a bilirubin through AhR pathway.

Bilirubin exhibits a novel anti-cancer effect on human adenocarcinoma.

Anti-oxidants are essential for intracellular free radical scavenging, as free radicals are one of the causes for tumorigenesis. Our objective was to use bilirubin and investigate its action on human carcinoma cell lines. Bilirubin manifested as a prooxidant showing its cytopathic effect on TMK-1, showing growth inhibition close to 50%. Cell cycle analysis showed an arrest at G0/G1. Flow cytometry investigations with Red CC-1 showed an increase by more than 2 times suggesting a prooxidative role of bilirubin. To check the effect of radicals on DNA, a Comet Assay displayed a typical comet's tail with bilirubin treated slides, only. Further, staining with DAPI showed apoptotic action of bilirubin. Decreased mitochondrial function by bilirubin was observed with Mitotracker Green FM staining. These unexpected data have led us to conclude that bilirubin has anti-cancer activity as a prooxidant and may have a more vital role in the human body than realized.

Bilirubin derived from heme degradation suppresses MHC class II expression in endothelial cells.

The enzymatic action of heme oxygenase (HO) is mediated by the cleavage of heme into carbon monoxide, ferrous iron, and biliverdin/bilirubin. Here, we show that induction of HO-1 expression, an inducible form of HO, down-regulates IFN-gamma-induced MHC class II expression in endothelial cells. Among three catalytic products of HO, bilirubin, but not carbon monoxide or ferrous iron, mediated the suppressive effects of HO through the reduction of mRNA levels of Stat-1-dependent class II transactivator. Expression of HO-1 could suppress the levels of IFN-gamma-induced Stat-1 phosphorylation. This effect could be mimicked by exposing the cells to one of its catalytic products, bilirubin. In addition, HO-1 or bilirubin could modulate the transcript activities of Stat-1-driven gene expression in luciferase reporter assays. These findings suggest an important role of HO-1 in the modulation of immune responses through suppression of MHC-II expression in antigen presenting cells. Our data provide a new line of evidence supporting HO-1-targeted therapy for immune modulation.

Bilirubin is highly effective in preventing in vivo delta-aminolevulinic acid-induced oxidative cell damage.

Delta-aminolevulinic acid (ALA), precursor of heme, accumulates in a number of organs, particularly in liver of patients with acute porphyrias or lead intoxication. This study characterizes the involvement of bilirubin as an antioxidant in a chronic intoxication with ALA. Female Wistar rats were injected intraperitoneally a daily dose of 40 mg ALA/body wt., during 10 days. A marked increase in lipid peroxidation and a decrease in GSH content were observed 24 h after the last injection of ALA. The activities of liver antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase were also diminished. ALA synthase (ALA-S) and heme oxygenase-1 were induced. Both ALA dehydratase (ALA-D) and porphobilinogenase (PBG-ase) activities were inhibited. Administration of bilirubin (5 mmol/kg body wt.) 2 h before ALA treatment entirely prevented the effects of ALA. Co-administration of ALA and Sn-protoporphyrin IX (Sn-PPIX; 100 microg/body wt., i.p.), a potent inhibitor of heme oxygenase, completely abolished its induction and provoked a marked decrease in liver GSH levels as well as an increase in lipid peroxidation. These results add further support to the proposal assigning bilirubin a key protective role against oxidative damage here induced by ALA.

Cholestatic effect of large bilirubin loads and cholestasis protection conferred by cholic acid co-infusion: a molecular and ultrastructural study.

BACKGROUND: Large intravenous bilirubin loads block biliary phospholipid secretion, produce canalicular membrane lesions and cause canalicular cholestasis. Cholic acid co-infusion forestalls these untoward effects. The aim of this study was first to determine whether bilirubin overload causes cholestasis through reducing the activity or the hepatic expression of the bile salt export pump (bsep) or Na-taurocholate co-transporting polypeptide (ntcp) and, secondly, whether cholic acid co-infusion forestalls cholestasis by upregulating bsep, ntcp or phosphoglycoprotein 3 (pgp3) expressions or activities. A further aim was to determine whether large bilirubin infusions also produce ultrastructural changes inside hepatocytes. METHODS: The effects of intravenous infusion of 2 g bilirubin over 150 min on hepatic expression of bsep, ntcp and pgp3 were studied in bile acid-depleted and cholic acid co-infused pigs, and related to canalicular bile acid transport and bile secretion. Effects on hepatocyte ultrastructural morphology were analysed by electron microscopy. RESULTS: Bilirubin-induced cholestasis reflected marked diminution of bsep and pgp3 transport activities and not reduced hepatic expression of these transporters. Hepatocyte ultrastructural abnormalities were predominantly confined to the hepatocyte canalicular membrane in cholestatic livers. Cholic acid co-infusion with bilirubin conferred complete cholestasis protection through enhancing pgp3 and bsep transporter activities and not through upregulating their expression. Bilirubin infusion did not change ntcp expression. CONCLUSION: Bilirubin-induced cholestasis is due to markedly impaired activity of the membrane-embedded bsep transporter consequent upon ultrastructural injury to the canalicular membrane. Cholic acid co-infusion with bilirubin enhances bsep and pgp3 activities and confers protection against canalicular membrane injury and bilirubin-induced cholestasis.

Effect of bilirubin in ischemia/reperfusion injury on rat small intestine.

PURPOSE: The aim of this study was to determine the effects of bilirubin in experimental small intestinal ischemia/reperfusion (I/R) injury in rats. METHODS: Thirty rats were divided into 5 groups (n = 6). In group S, saline and in group B, bilirubin, 20 mg/kg were infused via the jugular vein without an additional procedure. In groups S-IR, saline, B(1)-IR and B(2)-IR, 10 and 20 mg/kg/h of bilirubin were infused for 2 hours, respectively. In these groups, an I/R procedure was done after infusions by occluding the superior mesenteric artery for 45 minutes followed by 1 hour of reperfusion. After reperfusion, the small intestines were resected for histopathologic and malondialdehyde (MDA) assessments. Mucosal lesions were scored between 0 and 5. Malondialdehyde levels and histopathologic grades were analyzed statistically. RESULTS: Mucosal injury was severe in S-IR (grade 4 to 5), mild in B(1)-IR (grade 0 to 3) and none in B(2)-IR group (grade 0). Grades of group S-IR were higher than those of B(1)-IR and B(2)-IR statistically (P <.05). Tissue MDA levels of the S-IR group were significantly higher than those of B(1)-IR and B(2)-IR groups (U = 36, P <.05). Bilirubin levels correlated inversely with MDA levels (r = -0.94). CONCLUSIONS: Bilirubin effectively prevents intestinal I/R injury in rat. This observation is consistent with the hypotheses regarding bilirubin as an antioxidant, having a role in the body defense. J Pediatr Surg 36:1764-1767.