IL-1β and TNFα Differentially Influence NF-κB Activity and FasL-Induced Apoptosis in Primary Murine Hepatocytes During LPS-Induced Inflammation

Macrophage-derived cytokines largely influence the behavior of hepatocytes during an inflammatory response. We previously reported that both TNFα and IL-1β, which are released by macrophages upon LPS stimulation, affect Fas ligand (FasL)-induced apoptotic signaling. Whereas TNFα preincubation leads to elevated levels of caspase-3 activity and cell death, pretreatment with IL-1β induces increased caspase-3 activity but keeps cells alive. We now report that IL-1β and TNFα differentially influence NF-κB activity resulting in a differential upregulation of target genes, which may contribute to the distinct effects on cell viability. A reduced NF-κB activation model was established to further investigate the molecular mechanisms which determine the distinct cell fate decisions after IL-1β and TNFα stimulation. To study this aspect in a more physiological setting, we used supernatants from LPS-stimulated bone marrow-derived macrophages (BMDMs). The treatment of hepatocytes with the BMDM supernatant, which contains both IL-1β and TNFα, sensitized to FasL-induced caspase-3 activation and cell death. However, when TNFα action was blocked by neutralizing antibodies, cell viability after stimulation with the BMDM supernatant and FasL increased as compared to single FasL stimulation. This indicates the important role of TNFα in the sensitization of apoptosis in hepatocytes. These results give first insights into the complex interplay between macrophages and hepatocytes which may influence life/death decisions of hepatocytes during an inflammatory reaction of the liver in response to a bacterial infection

Genome-wide comparison between IL-17 and combined TNF-alpha/IL-17 induced genes in primary murine hepatocytes

<p>Abstract</p> <p>Background</p> <p>Cytokines such as TNF-alpha and IL-1beta are known for their contribution to inflammatory processes in liver. In contrast, the cytokine IL-17 has not yet been assigned a role in liver diseases. IL-17 can cooperate with TNF-alpha to induce a synergistic response on several target genes in different cell lines, but no data exist for primary hepatocytes. To enhance our knowledge on the impact of IL-17 alone and combined with TNF-alpha in primary murine hepatocytes a comprehensive microarray study was designed. IL-1beta was included as this cytokine is suggested to act in a similar manner as the combination of TNF-alpha and IL-17, especially with respect to its role in mRNA stabilization.</p> <p>Results</p> <p>The present microarray analysis demonstrates that primary murine hepatocytes responded to IL-17 stimulation by upregulation of chemokines and genes, which are functionally responsible to increase and sustain inflammation. Cxcl2, Nfkbiz and Zc3h12a were strongly induced, whereas the majority of the genes were only very moderately up-regulated. Promoter analysis revealed involvement of NF-kappaB in the activation of many genes. Combined stimulation of TNF-alpha/IL-17 resulted in enhanced induction of gene expression, but significantly synergistic effects could be applied only to a few genes, such as Nfkbiz, Cxcl2, Zc3h12 and Steap4. Comparison of the gene expression profile obtained after stimulation of TNF-alpha/IL-17 versus IL-1beta proposed an "IL-1beta-like effect" of the latter cytokine combination. Moreover, evidence was provided that modulation of mRNA stability may be a major mechanism by which IL-17 regulates gene expression in primary hepatocytes. This assumption was exemplarily proven for Nfkbiz mRNA for the first time in hepatocytes. Our studies also suggest that RNA stability can partially be correlated to the existence of AU rich elements, but further mechanisms like the RNase activity of the up-regulated Zc3h12a have to be considered.</p> <p>Conclusions</p> <p>Our microarray analysis gives new insights in IL-17 induced gene expression in primary hepatocytes highlighting the crosstalk with the NF-kappaB signaling pathway. Gene expression profile suggests IL-17 alone and in concert with TNF-alpha a role in sustaining liver inflammatory processes. IL-17 might exceed this function by RNA stabilization.</p

Isolation of Flavonoids from Deguelia duckeana and Their Effect on Cellular Viability, AMPK, eEF2, eIF2 and eIF4E

Preparations of Deguelia duckeana, known in Brazil as timbó, are used by indigenous people to kill fish. Reinvestigation of its extracts resulted in the isolation and identification of 11 known flavonoids identified as 3,5,4’-trimethoxy-4-prenylstilbene (1), 4-methoxyderricidine (2), lonchocarpine (3), 4-hydroxylonchocarpine (4), 4-methoxylonchocarpine (5), 5-hydroxy-4’,7-dimethoxy-6-prenylflavanone (6), 4’-hydroxyisolonchocarpine (7), 4’-methoxyisolonchocarpine (8), 3’,4’,7-trimethoxyflavone (9), 3’,4’-methylenedioxy-7-methoxyflavone (10), and 2,2-dimethyl-chromone-5,4’-hydroxy-5’-methoxyflavone (11). Except for 1, 3, and 4 all of these flavonoids have been described for the first time in D. duckeana and the flavanone 6 for the first time in nature. Compounds 2, 3, 4, 7, 9, and 10 were studied for their potential to induce cell death in neuronal SK-N-SH cells. Only the chalcone 4 and the flavanone 7 significantly induced lactate dehydrogenase (LDH) release, which was accompanied by activation of caspase-3 and impairment of energy homeostasis in the MTT assay and may explain the killing effect on fish. Interestingly, the flavone 10 reduced cell metabolism in the MTT assay without inducing cytotoxicity in the LDH assay. Furthermore, the flavonoids 2, 3, 4, 7, and 10 induced phosphorylation of the AMP-activated protein kinase (AMPK) and the eukaryotic elongation factor 2 (eEF2). The initiation factor eIF4E was dephosphorylated in the presence of these compounds. The initiation factor eIF2alpha was not affected. Further studies are needed to elucidate the importance of the observed effects on protein synthesis and potential therapeutic perspectives.Conselho Nacional de Desenvolvimento Científico e Tecnológico/[]/CNPq/BrazilAlbert Ludwigs University Freiburg///GermanyGerman Research Foundation//DFG/GermanyUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA

Modeling the TNFα-Induced Apoptosis Pathway in Hepatocytes

The proinflammatory cytokine TNFα fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Recently, we showed that TNFα is able to sensitize primary murine hepatocytes cultured on collagen to Fas ligand-induced apoptosis and presented a mathematical model of the sensitizing effect. Here, we analyze how TNFα induces apoptosis in combination with the transcriptional inhibitor actinomycin D (ActD). Accumulation of reactive oxygen species (ROS) in response to TNFR activation turns out to be critical for sustained activation of JNK which then triggers mitochondrial pathway-dependent apoptosis. In addition, the amount of JNK is strongly upregulated in a ROS-dependent way. In contrast to TNFα plus cycloheximide no cFLIP degradation is observed suggesting a different apoptosis pathway in which the Itch-mediated cFLIP degradation and predominantly caspase-8 activation is not involved. Time-resolved data of the respective pro- and antiapoptotic factors are obtained and subjected to mathematical modeling. On the basis of these data we developed a mathematical model which reproduces the complex interplay regulating the phosphorylation status of JNK and generation of ROS. This model was fully integrated with our model of TNFα/Fas ligand sensitizing as well as with a published NF-κB-model. The resulting comprehensive model delivers insight in the dynamical interplay between the TNFα and FasL pathways, NF-κB and ROS and gives an example for successful model integration

Current knowledge on metabolism of flavonoids. Part 2. Resorption and metabolism of flavones, flavanones, flavanes, proanthocyanidines, and isoflavonoids

A review with 57 refs. is given including ring cleavage products and other metabolites, resorption and metab. of intact compds., and pharmacokinetic aspects of the title compds

Current knowledge on the metabolism of flavonoids. Part 1. Resorption and metabolism of flavonols

A review with 62 refs. is given on resorption, metab., and pharmacokinetics of flavonols including a crit. assessment. Ring cleavage and metabolization, resorption and metabolization of intact compds., and pharmacokinetic points of view were discussed. Within the resorption possibilities of flavonols from the food, the metabolization by intestinal bacteria plays the main role. Flavonoid metabolites are the real bioavailable compds

Radical scavenger activity of different 3',4'-dihydroxyflavonols and 1,5-dicaffeoylquinic acid studied by inhibition of chemiluminescence

To gain more insights into structure-activity relationships, four 3',4'-dihydroxyflavonols differing in the substitution of the A and C rings and 1,5-dicaffeoylquinic acid were evaluated for their ability to inhibit chemiluminescence of human neutrophils stimulated with opsonized zymosan or FMLP as well as in an enzymic system with H2O2 and horseradish peroxidase. It could be shown that an addnl. o-dihydroxy structure in the A-ring, or a 6-methoxy group, resp., has no significant influence, thus confirming the o-dihydroxy group of the B-ring as the most important structural feature for the radical scavenger activity. It can be supposed that the main effect of the tested flavonols is based on their inhibition of myeloperoxidase, besides inhibition of enzymes involved in activating the NADPH-oxidase, and a direct reaction with oxygen radicals. Inhibition of chemiluminescence by 1,5-dicaffeoylquinic acid was in the same order as those obsd. with the flavonols

Diterpenes and sesquitrpenes from Mikania banisteriae

Two new ent-kaurane diterpenes, 18,19-diacetoxy-ent-kaur-16-ene and 17-oxo-ent-kaur-15(16)-en-18-oic acid, were isolated from the aerial parts of Mikania banisteriae, together with the known ent-kauranes ent-kaur-16-en-18-oic acid and ent-kaur-16-en-18-ol. The structures were established by mass spectrometry, 1H NMR and partly 13C NMR spectroscopy. In addition, the sesquiterpene lactones eudesma-4(15),7(11)-dien-8β,12-olide and eudesma-4(15),7(11),8(9)-trien-12-olide as well as the sesquiterpenes 1β,6α-dihydroxyeudesm-4(15)-ene and caryophylleno-xide were identified by direct comparison (TLC, GC, GC-MS) with authentic samples. The chemotaxonomic significance is briefly discussed. ent-Kaur-16-en-18-oic acid and ent-kaur-16-en-19-oic acid showed no antifeedant, antimicrobial and antiinflammatory properties.Institut für Pharmazeutische Chemie//IPC/AlemaniaUniversität Düsseldorf//HHU/AlemaniaUniversidad Nacional//UNA/Costa RicaInstitut für Pharmazeutische Biologie//IPB/AlemaniaUniversity of Würzburg//JMU/AlemaniaShanghai Institute of Materia Medica///ChinaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic