Differentiation of the human liver progenitor cell line (HepaRG) on a microfluidic-based biochip.

HepaRG is a bipotent stem cell line that can be differentiated towards hepatocyte-like and biliary-like cells. The entire cultivation process requires 1 month and relies on the addition of 2% dimethyl sulfoxide (DMSO) to the culture. Our motivation in this research is to differentiate HepaRG cells (progenitor cells and undifferentiated cells) towards hepatocyte-like cells by minimizing the cultivation time and without using DMSO treatment by instead using a microfluidic device combined with the following strategies: (a) comparison of extracellular matrices (matrigel and collagen I), (b) types of flow (one or both sides), and (c) effects of DMSO. Our results demonstrate that matrigel promotes the differentiation of progenitor cells towards hepatocytes and biliary-like cells. Moreover, the frequent formation of HepaRG cell clusters was observed by a supply of both sides of flow, and the cell viability and liver specific functions were influenced by DMSO. Finally, differentiated HepaRG progenitor cells cultured in a microfluidic device for 14 days without DMSO treatment yielded 70% of hepatocyte-like cells with a highly polarized organization that reacted to stimulation with IL-6 to produce C-reactive protein (CRP). This culture model has high potential for investigating cell differentiation and liver pathophysiology research.

Study of melatonin-mediated effects on various hepatic inflammatory responses stimulated by IL-6 in a new HepG2-on-a-chip platform.

Hepatocytes exhibit diverse reactions upon stimulation with the interleukin IL-6, mainly in the context of inflammation and energy metabolism. Melatonin has been shown to exert pleiotropic protective actions, such as anti-inflammation and anti-oxidative stress on many cell- and organ-types. The key role of the liver to maintain homeostasis and metabolic regulation prompted us to evaluate the direct modification of IL-6-induced alterations in HepG2 cells in a chip by melatonin. IL-6 administration was followed by the reduced expression and activity of MRP2, a loss of CYP1A activity, and the decline of PXR expression. Other effects were the induction of acute phase responses (reduced albumin production as well as increased CRP and hepcidin expression) and lowered expression of CREB3L3. IL-6 affected also the mitochondrial membrane potential together with elevated mitochondrial superoxide generation, and glycogen deposition was reduced. Melatonin counteracted all observed IL-6-induced alterations except the rise in CRP release and CYP1A activity. Altogether, this new in vitro model can be applied to investigate hepatic inflammatory responses stimulated by IL-6, and these results indicate that hepatocellular inflammatory responses to IL-6 are mitigated by melatonin.

Metabolism of 3-pentanone under inflammatory conditions.

Breath analysis of rats using multi-capillary column ion-mobility spectrometry (MCC-IMS) revealed alterations in acetone and other ketones, including 3-pentanone, during inflammation. The alterations seem likely to result from oxidative branched-chain keto acid (BCKA) catabolism. We therefore tested the hypothesis that 3-pentanone arises during inflammation from increased BCKA oxidation in the liver with consequent accumulation of propionyl-CoA and its condensation products. Male Sprague-Dawley rats were anaesthetised and ventilated for 24 h or until death. Exhaled breath was analysed by MCC-IMS while rats were injected with low and high doses of lipopolysaccharide (LPS), tumour necrosis factor α (TNFα), or vehicle. The exhaled 3-pentanone peak was identified by pure substance measurements. Blood was collected 12 h after treatment for the determination of cytokine concentrations; transcription enzymes for BCKA catabolism and the activity of the BCKA dehydrogenase were analysed in liver tissue by quantitative real-time PCR and western blotting. Exhaled 3-pentanone decreased in all groups, but minimum concentrations with high-dose LPS (0.24 ± 0.31 volts; mean ± SD), low-dose TNFα (0.17 ± 0.10 volts) and high-dose TNFα (0.13 ± 0.04 volts) were lower than in vehicle animals (0.27 ± 0.12 volts). At 60% and 85% survival times (svt) concentrations of exhaled 3-pentanone increased significantly in all animals treated with low-dose LPS, (svt60% 0.38 ± 0.18 volts, svt85% 0.62 ± 0.15 volts) and high-dose LPS (0.26 ± 0.28 volts, 0.40 ± 0.22 volts), as well as low-dose TNFα, (0.20 ± 0.09 volts, 0.39 ± 0.17 volts) and high-dose TNFα (0.18 ± 0.06 volts, 0.34 ± 0.08 volts), but not in vehicle rats (0.27 ± 0.12 volts, 0.30 ± 0.09 volts). BCKA catabolism was seen in the liver, with increased expression and activity of the branched-chain alpha-keto acid dehydrogenase (BCKD), lower expression of the propionyl-CoA carboxylase (PCC) subunits, and altered expression levels of BCKD regulating enzymes. Exhaled 3-pentanone may arise from altered BCKA catabolism. Our results suggest that excessive propionyl-CoA is possibly generated from BCKAs via increased activity of BCKD, but may undergo unusual condensation reactions rather than being physiologically processed to methylmalonyl-CoA by PCC. The pattern of 3-pentanone during early and prolonged inflammation suggests that reuse of BCKAs for the synthesis of new proteins might be initially favoured. As inflammatory conditions persist, substrates for cellular energy supply are required which activate irreversible degradation of excessive BCKA to propionyl-CoA yielding increased levels of exhaled 3-pentanone.

Melatonin or ramelteon therapy differentially affects hepatic gene expression profiles after haemorrhagic shock in rat--A microarray analysis.

BACKGROUND & AIMS: Melatonin has been demonstrated to reduce liver damage in different models of stress. However, there is only limited information on the impact of this hormone on hepatic gene expression. The aim of this study was, to investigate the influence of melatonin or the melatonergic agonist ramelteon on hepatic gene expression profiles after haemorrhagic shock using a whole genome microarray analysis. METHODS: Male Sprague-Dawley rats (200-300 g, n=4/group) underwent haemorrhagic shock (mean arterial pressure 35±5 mmHg). After 90 min of shock, animals were resuscitated with shed blood and Ringer's and treated with vehicle (5% dimethyl sulfoxide), melatonin or ramelteon (each 1.0 mg/kg intravenously). Sham-operated animals were treated likewise but did not undergo haemorrhage. After 2 h of reperfusion, the liver was harvested, and a whole genome microarray analysis was performed. Functional gene expression profiles were determined using the Panther® classification system; promising candidate genes were evaluated by quantitative polymerase chain reaction (PCR). RESULTS: Microarray and PCR data showed a good correlation (r(2)=0.84). A strong influence of melatonin on receptor mediated signal transduction was revealed using the functional gene expression profile analysis, whereas ramelteon mainly influenced transcription factors. Shock-induced upregulation of three candidate genes with relevant functions for hepatocytes (ppp1r15a, dusp5, rhoB) was significantly reduced by melatonin (p<0.05 vs. shock/vehicle), but not by ramelteon. Two genes previously known as haemorrhage-induced (il1b, s100a8) were transcriptionally repressed by both drugs. CONCLUSIONS: Melatonin and ramelteon appear to induce specific hepatic gene expression profiles after haemorrhagic shock in rats. The observed differences between both substances are likely to be attributable to a distinct mechanism of action in these agents.

Impact of melatonin receptor deletion on intracellular signaling in spleen cells of mice after polymicrobial sepsis.

OBJECTIVE: Melatonin is known to influence immune functions and to ameliorate outcome after septic challenge but it is unknown whether this is mediated by melatonin receptor activation. This study aimed to elucidate molecular differences in spleen and ex vivo splenocytes of wild-type (WT) and melatonin receptor double knockout mice (KO) after polymicrobial sepsis. SUBJECTS AND METHODS: C3H/HeN wild-type and MT1-/-/MT2-/- mice underwent sham operation or cecum ligation and incision (CLI) and remained anesthetized for 1 h. Splenocytes were isolated and treated in culture with physiological melatonin concentrations (1 nM). RESULTS: Plasma TNFα levels were consistently high after 1 h of CLI. Basal circulating leukocyte numbers were slightly higher in KO animals. We detected transcriptional differences in splenocytes of the knockout strain concerning proinflammatory mediators. Expression levels of IL-1ß, IL-2, CXCR2, L-Selectin, TNFα, CXCL2 and ICAM-1 were strongly increased in splenocytes of KO mice. Splenocytes of KO mice displayed reduced ERK and p38 as well as increased JNK phosphorylation. None of the analyzed factors were influenced by melatonin in the culture medium. CONCLUSIONS: The results of this study indicate an increased proinflammatory status of mice deficient in both membrane-bound melatonin receptors reflected by altered activation of MAPK cascades and transcriptional activation of proinflammatory mediators.

Melatonin receptors mediate improvements of survival in a model of polymicrobial sepsis.

OBJECTIVES: Melatonin has been demonstrated to improve survival after experimental sepsis via antioxidant effects. Yet, recent evidence suggests that this protective capacity may also rely on melatonin receptor activation. Therefore, the present study was designed to investigate whether selective melatonin receptor-agonist ramelteon may influence survival and immune response in a model of polymicrobial sepsis in rats, wild-type and melatonin receptor MT1/MT2 double knockout mice. DESIGN: Prospective, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats (200-250 g) and male C3H/HeN wild-type and MT1/MT2 receptor knockout mice (20-22 g). INTERVENTIONS: Animals underwent cecal ligation and incision and remained anesthetized for evaluation of survival for 12 hours (rats: n = 15 per group) or 15 hours (mice: n = 10 per group). Analysis of immune response by means of enzyme-linked immunosorbent assay was performed before and 5 hours after cecal ligation and incision (rats only; n = 5 per group). After induction of sepsis, animals were treated IV with vehicle, different doses of melatonin (rats: 0.01/0.1/1.0/10 mg/kg; mice: 1.0 mg/kg), ramelteon, melatonin receptor-antagonist luzindole, ramelteon + luzindole, or melatonin + luzindole (each 1.0 mg/kg). Sham controls underwent laparotomy but not cecal ligation and incision. MEASUREMENTS AND MAIN RESULTS: Compared with vehicle, administration of ramelteon or melatonin significantly improved median survival time in rats (sepsis/melatonin [0.1 mg/kg], 554 min, [1.0 mg/kg] 570 min, [10 mg/kg] 579 min; sepsis/ramelteon, 468 min; each p < 0.001 vs sepsis/vehicle, 303 min) and wild-type mice (sepsis/melatonin, 781 min; sepsis/ramelteon, 701 min; both p < 0.001 vs sepsis/vehicle, 435 min). This effect was completely antagonized by coadministration of luzindole in all groups. Melatonin, ramelteon, or luzindole had no significant effect on survival time in knockout mice. Significantly elevated concentrations of tumor necrosis factor-α, interleukin-6, and interleukin-10 were observed 5 hours after cecal ligation and incision in rats (p < 0.05 vs baseline and corresponding sham); neither ramelteon nor melatonin treatment significantly affected immune response. CONCLUSIONS: Melatonin receptors mediate improvements of survival after polymicrobial sepsis in rats and mice; this effect appears to be independent from major alterations of cytokine release.