Primary Liver Cell Cultures Research Articles

Nocturnal melatonin increases glucose uptake via insulin-independent action in the goldfish brain.

Melatonin, a neurohormone nocturnally produced by the pineal gland, is known to regulate the circadian rhythm. It has been recently reported that variants of melatonin receptors are associated with an increased risk of hyperglycemia and type 2 diabetes, suggesting that melatonin may be involved in the regulation of glucose homeostasis. Insulin is a key hormone that regulates circulating glucose levels and cellular metabolism after food intake in many tissues, including the brain. Although cells actively uptake glucose even during sleep and without food, little is known regarding the physiological effects of nocturnal melatonin on glucose homeostasis. Therefore, we presume the involvement of melatonin in the diurnal rhythm of glucose metabolism, independent of insulin action after food intake. In the present study, goldfish (Carassius auratus) was used as an animal model, since this species has no insulin-dependent glucose transporter type 4 (GLUT4). We found that in fasted individuals, plasma melatonin levels were significantly higher and insulin levels were significantly lower during the night. Furthermore, glucose uptake in the brain, liver, and muscle tissues also significantly increased at night. After intraperitoneal administration of melatonin, glucose uptake by the brain and liver showed significantly greater increases than in the control group. The administration of melatonin also significantly decreased plasma glucose levels in hyperglycemic goldfish, but failed to alter insulin mRNA expression in Brockmann body and plasma insulin levels. Using an insulin-free medium, we demonstrated that melatonin treatment increased glucose uptake in a dose-dependent manner in primary cell cultures of goldfish brain and liver cells. Moreover, the addition of a melatonin receptor antagonist decreased glucose uptake in hepatocytes, but not in brain cells. Next, treatment with N1-acetyl-5-methoxykynuramine (AMK), a melatonin metabolite in the brain, directly increased glucose uptake in cultured brain cells. Taken together, these findings suggest that melatonin is a possible circadian regulator of glucose homeostasis, whereas insulin acquires its effect on glucose metabolism following food intake.

High Phenotypic Variation between an In Vitro-Passaged Fowl Adenovirus Serotype 1 (FAdV-1) and Its Virulent Progenitor Strain despite Almost Complete Sequence Identity of the Whole Genomes.

Adenoviral gizzard erosion is an emerging disease with negative impact on health and production of chickens. In this study, we compared in vitro and in vivo characteristics of a fowl adenovirus serotype 1 (FAdV-1), attenuated by 53 consecutive passages in primary chicken embryo liver (CEL) cell cultures (11/7127-AT), with the virulent strain (11/7127-VT). Whole genome analysis revealed near-complete sequence identity between the strains. However, a length polymorphism in a non-coding adenine repeat sequence (11/7127-AT: 11 instead of 9) immediately downstream of the hexon open reading frame was revealed. One-step growth kinetics showed delayed multiplication of 11/7127-AT together with significantly lower titers in cell culture (up to 4 log10 difference), indicating reduced replication efficiency in vitro. In vivo pathogenicity and immunogenicity were determined in day-old specific pathogen-free layer chicks inoculated orally with the respective viruses. In contrast to birds infected with 11/7127-VT, birds infected with 11/7127-AT did not exhibit body weight loss or severe pathological lesions in the gizzard. Virus detection rates, viral load in organs and virus excretion were significantly lower in birds inoculated with 11/7127-AT. Throughout the experimental period, these birds did not develop measurable neutralizing antibodies, prevalent in birds in response to 11/7127-VT infection. Differences in pathogenicity between the virulent FAdV-1 and the attenuated strain could not be correlated to prominently discriminate genomic features. We conclude that differential in vitro growth profiles indicate that attenuation is linked to modulation of viral replication during interaction of the virus with the host cells. Thus, hosts would be unable to prevent the rapid replication of virulent FAdV leading to severe tissue damage, a phenomenon broadly applicable to further FAdV serotypes, considering the substantial intra-serotype virulence differences of FAdVs and the variation of diseases.

Insulin-dependent glucose consumption dynamics in 3D primary human liver cultures measured by a sensitive and specific glucose sensor with nanoliter input volume.

The liver plays a central role in glucose homeostasis and hepatic insulin resistance constitutes a key feature of type 2 diabetes. However, platforms that accurately mimic human hepatic glucose disposition and allow for rapid and scalable quantification of glucose consumption dynamics are lacking. Here, we developed and optimized a colorimetric glucose assay based on the glucose oxidase-peroxidase system and demonstrate that the system can monitor glucose consumption in 3D primary human liver cell cultures over multiple days. The system was highly sensitive (limit of detection of 3.5µM) and exceptionally accurate (R2 =0.999) while requiring only nanoliter input volumes (250nL), enabling longitudinal profiling of individual liver microtissues. By utilizing a novel polymer, off-stoichiometric thiol-ene (OSTE), and click-chemistry based on thiol-Michael additions, we furthermore show that the assay can be covalently bound to custom-build chips, facilitating the integration of the sensor into microfluidic devices. Using this system, we find that glucose uptake of our 3D human liver cultures closely resembles human hepatic glucose uptake in vivo as measured by euglycemic-hyperinsulinemic clamp. By comparing isogenic insulin-resistant and insulin-sensitive liver cultures we furthermore show that insulin and extracellular glucose levels account for 55% and 45% of hepatic glucose consumption, respectively. In conclusion, the presented data show that the integration of accurate and scalable nanoliter glucose sensors with physiologically relevant organotypic human liver models enables longitudinal profiling of hepatic glucose consumption dynamics that will facilitate studies into the biology and pathobiology of glycemic control, as well as antidiabetic drug screening.

Glycogene Expression Profiling of Hepatic Cells by RNA-Seq Analysis for Glyco-Biomarker Identification.

Glycans are primarily generated by “glycogenes,” which consist of more than 200 genes for glycosynthesis, including sugar-nucleotide synthases, sugar-nucleotide transporters, and glycosyltransferases. Measuring the expression level of glycogenes is one of the approaches to analyze the glycomes of particular biological and clinical samples. To develop an effective strategy for identifying the glycosylated biomarkers, we performed transcriptome analyses using quantitative real-time polymerase chain reaction (qRT-PCR) arrays and RNA sequencing (RNA-Seq). First, we measured and analyzed the transcriptome from the primary culture of human liver cells and hepatocarcinoma cells using RNA-Seq. This analysis revealed similar but distinctive expression profiles of glycogenes among hepatic cells as indicated by the qRT-PCR arrays, which determined a copy number of 186 glycogenes. Both data sets indicated that altered expression of glycosyltransferases affect the glycosylation of particular glycoproteins, which is consistent with the mass analysis data. Moreover, RNA-Seq analysis can uncover mutations in glycogenes and search differently expressed genes out of more than 50,000 distinct human gene transcripts including candidate biomarkers that were previously reported for hepatocarcinoma cells. Identification of candidate glyco-biomarkers from the expression profile of the glycogenes and proteins from liver cancer tissues available from public database emphasized the possibility that even though the expression level of biomarkers might not be altered, the expression of the glycogenes modifying biomarkers, generating glyco-biomarkers, might be different. Pathway analysis revealed that ~20% of the glycogenes exhibited different expression levels in normal and cancer cells. Thus, transcriptome analyses using both qRT-PCR array and RNA-Seq in combination with glycome and glycoproteome analyses can be advantageous to identify “glyco-biomarkers” by reinforcing information at the expression levels of both glycogenes and proteins.

1859-P: Hepatic NIK Augments Hepatocyte Ferroptosis and Hepatic Inflammation in Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of obesity and diabetes. Hepatocyte injury and death pathologically mark a turning point from simple liver steatosis to nonalcoholic steatohepatitis (NASH). Chronic hepatocyte injury and hepatic inflammation drive liver fibrosis, leading to liver cirrhosis, end-stage liver failure, and/or hepatocellular carcinoma. We previously reported that NAFLD is associated with a marked elevation of NF-κB-inducing kinase (NIK) in the liver. NIK in turn augments the hepatic hyperglycemic response to counterregulatory hormone glucagon, contributing to hyperglycemia and diabetes progression. Here, we demonstrate that hepatic NIK also promotes hepatocyte injury and death, exacerbating liver disease. We found that hepatocyte-specific overexpression of NIK substantially increased hepatocyte death and liver inflammation in mice treated with acetaminophen (APAP), a hepatocyte-toxic drug. Conversely, hepatocyte-specific deletion of NIK significantly blunted APAP-induced liver injury and hepatitis. In line with these findings, in primary liver cell cultures, overexpression of NIK substantially enhanced APAP sensitivity to induce cell death. Notably, NIK overexpression dramatically increased liver reactive oxygen species (ROS) levels in APAP-treated mice, and ablation of hepatocyte NIK had the opposite effects. Treatment with antioxidant N-acetylcysteine abrogated the ability of NIK and APAP to induce hepatocyte death and liver inflammation. Given that ROS drives ferroptosis, we treated primary liver cells with ferroptosis inhibitors ferrostatin 1 and liproxtatin-1. Both ferrostatin 1 and liproxtatin-1 dose-dependently suppressed NIK/APAP-induced cell death. Collectively, our data suggest that aberrant activation of hepatic NIK augments hepatocyte ferroptosis, thereby promoting liver injury, liver inflammation, and liver disease progression. Disclosure X. Zhong: None. L. Rui: None.

Oxidative stress in liver cell culture from mullet, Liza klunzingeri, induced by short-term exposure to benzo[a]pyrene and nonylphenol.

The present investigation aimed to use primary liver cell culture obtained from mullet, Liza klunzingeri, to evaluate the toxic effects of benzo[a]pyrene (BaP) and nonylphenol (NP) on the antioxidant defense system. Liver samples taken from 20 L. klunzingeri were digested with 0.1% collagenase IV. The digested cells were then moved to Leibovitz L-15 culture medium and incubated at 25°C for 2weeks. 10-5mol/l of BaP and 10-4mol/l of NP were considered as the half maximal inhibitory concentration (IC50). Cells were then incubated with L-15 medium containing BaP (0[control], 10-6,2× 10-6,3× 10-6mol/l) and NP (0[control],10-5,2× 10-5,3× 10-5mol/l), and sampling was performed after 6, 12, and 24h of incubation for measurement of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), lipid peroxidation (LPO), total antioxidant power, and total protein. The lowest concentration of BaP and NP did not have considerable toxic effects on cultivated hepatocytes. The activities of SOD, CAT, GPx, LPO, total antioxidant power, and total protein changed dose-dependently in cells treated with BaP and NP. In conclusion, based on the results, short-term exposure to BaP and NP induced the oxidative stress in cultivated liver cells of L. klunzingeri. The toxicity of both pollutants is mainly because of the induction of the reactive oxygen species (ROS), which lead to cell membrane disruption, damage of cellular metabolism, and interference with cellular macromolecules.

Effects of the sulfated polysaccharide ulvan from Ulva ohnoi on the modulation of the immune response in Senegalese sole (Solea senegalensis)

Sulfated polysaccharides derived from green seaweeds exhibit many beneficial biological activities and have great potential to be used as nutraceutical in aquaculture. In this work, we evaluated the effects of the sulfated polysaccharide ulvan from Ulva ohnoi on Senegalese sole (Solea senegalensis) juveniles at the transcriptomic level. Cytotoxicity assay performed in liver primary cell cultures from sole determined that the different ulvan concentrations assayed did not impair cell viability. Juveniles were intraperitoneally (IP) injected with ulvan (0.5 mg/fish) followed by a challenge with Photobacterium damselae subsp. piscicida (Phdp) at 7 days. RNASeq analyses at 2 days post injection (dpi) revealed that 402 transcripts were differentially expressed in liver between ulvan IP injected and control groups before the challenge. Genes related to bacterial and antiviral defence, complement system, chemokines, proteasomes and antigen presentation were upregulated in ulvan treated groups. A detailed expression analysis of sixteen genes related to innate and adaptive immune system was performed in two systemic tissues: liver and spleen. Ulvan injection provoked the upregulation of tlr22 and a transient inflammatory response was initiated in both liver and spleen at 2 dpi. As consequence, expression of acute phase proteins, antimicrobial peptides and complement genes was induced. Moreover, expression of mhcI, mhcII, psmb10 and bcl6 was also induced 2 dpi. At 2 dpi with Phdp, inflammatory cytokines and genes related to bacterial and antiviral defense, iron metabolism, complement system and antigen presentation were differentially modulated in survival juveniles previously IP injected with ulvan. Moreover, mortality was retarded in ulvan treated juveniles. These results provide new evidence about the role of ulvan as a bioactive compound with immunomodulatory activity in Senegalese sole as well as its possible use as vaccine adjuvant against Phdp. This is the first published study that evaluates the transcriptomic response of Senegalese sole IP injected with ulvan.

Exposure of liver cell culture from the orange-spotted grouper, Epinephelus coioides, to benzo[a]pyrene and light results in oxidative damage as measured by antioxidant enzymes

Among the various toxicants discharged into aquatic environments, benzo (a) pyrene (BaP) has been shown to effect on the antioxidant system of fish and the evaluation of its impact on biota is of considerable concern. The aim of the present study was to use the primary hepatocyte culture obtained from the orange-spotted grouper, Epinephelus coioides, to evaluate the adverse effects of benzo (a) pyrene (BaP) on cell viability and liver antioxidant system. BaP was selected for its high ability to produce reactive oxygen species (ROS) and oxidative stress. The liver was minced by a scalpel and digested in the PBS solution with 0.1% collagenase IV at room temperature for 20 min. Then, the cell suspension was transferred to a plate contained an equal amount of Leibovitz's L-15 medium with 20% fetal bovine serum (FBS), 100 IU mL−1 of penicillin and 100 μg mL−1 streptomycin. 5 mL of cell suspension were plated into sterile 25 cm2 tissue culture flasks at the density of 1.5 × 106 cell/ml L-15 and incubated at 30 °C for two weeks. The medium was renewed after 24–48 h. The number of the liver cells was adjusted to 4 × 106 after two weeks. 10−4 mol l−1 was verified by MTT assay as the IC50 of BaP. Then, hepatocytes were exposed to three concentrations of BaP (10−5, 2 × 10−5, 3 × 10−5 mol L−1) and incubated for 24 h. Samples were collected after 6, 12 and 24 h and the amounts of SOD, CAT, GPx, LPO, LDH, AST, ALT, ALP and total protein were analyzed. The results showed that, 10−5 mol L−1 of BaP was not significantly toxic to cultivated hepatocytes, however, the sensitivity of cells to BaP increased in a dose-related pattern. The activity of the antioxidant enzymes (SOD, CAT and GPx) and liver enzymes (ALT, AST, ALP, LDH) significantly increased, though the amount of LPO, total antioxidant power and total protein decreased dose-dependently in BaP-exposed cells. In conclusion, according to the finding of the present study, BaP has a high potential to induce the oxidative stress in primary liver cell culture of E. coioides.

Synthesis, Photochemical and In Vitro Cytotoxic Evaluation of New Iodinated Aminosquaraines as Potential Sensitizers for Photodynamic Therapy.

In this work, several benzothiazole-based aminosquaraine dyes, displaying strong absorption within the so-called phototherapeutic window (650–800 nm), were synthesized. The ability, of all the new dyes, to generate singlet oxygen was assessed by determining the correspondent phosphorescence emission and through the comparison with a standard. The quantum yields of singlet oxygen generation were determined and exhibited to be strongly dependent on the nature of the amino substituents introduced in the squaric ring. The photodynamic activity of the synthesized dyes was tested against four human tumor cell lines: breast (MCF-7), lung (NCI-H460), cervical (HeLa) and hepatocellular (HepG2) carcinomas; and a non-tumor porcine liver primary cell culture (PLP2). All the compounds synthesized were found to be able to inhibit tumor cells growth upon irradiation more than in the dark, in most of the cases, very significantly. Considering the photodynamic activity exhibited and the low toxicity displayed for the non-tumor cells, some of the synthetized dyes can be regarded as potential candidates as photosensitizers for PDT.

Cloning, expression prolife, and immune characterization of a novel stat family member (stat5bl) in Chinese tongue sole (Cynoglossus semilaevis)

STAT plays important roles in innate immunity during JAK/STAT signaling pathway, and STAT5 is particularly focused due to the existence of duplicated forms in fish and mammal. In Chinese tongue sole, stat5bl was suggested to be a candidate related to Vibrio harveyi resistance based on previous QTL screening. In this study, the full length of stat5bl cDNA was cloned and its expression patterns were analyzed. stat5bl was predominantly expressed in immune tissues, where the highest level was observed in liver, followed by skin and gill. Time course expression patterns were examined in six tissues (liver, skin, gill, kidney, intestine, spleen) after V. harveyi infection. stat5bl could be up-regulated by V. harveyi infection in all tissues except liver, despite the timepoints of peak were different. In contrast, stat5bl was significantly downregulated in liver. To elucidate the role of stat5bl in liver, in vitro RNAi were performed using primary liver cell culture. Knockdown of stat5bl could regulate the expression of genes closely related to JAK/STAT pathway. This study would enlarge our understanding of stat5bl in fish immunity.

Hypersensitive assessment of aryl hydrocarbon receptor transcriptional activity using a novel truncated cyp1a promoter in zebrafish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant (POP), an unintentional byproduct of various industrial processes, and a human carcinogen. The expression of the cytochrome P450 1A (cyp1a) gene is upregulated in the presence of TCDD through activating the aryl hydrocarbon receptor pathway in a dose-dependent manner. Several essential response elements, including the 8 potential xenobiotic response elements in the cyp1a promoter region, have been identified to be the main functional parts for the response to TCDD. Thus, we aimed to develop a convenient and sensitive biomonitoring tool to examine the level of POPs in the environment and evaluate its potential human health risks by TCDD. Here, we established a transgenic zebrafish model with a red fluorescent reporter gene ( mCherry) using the truncated cyp1a promoter. Under exposure to TCDD, the expression pattern of mCherry in the reporter zebrafish mirrored that of endogenous cyp1a mRNA, and the primary target tissues for TCDD were the brain vessels, liver, gut, cloaca, and skin. Our results indicated that exposure of the embryos to TCDD at concentrations as low as 0.005 nM for 48 h, which did not elicit morphologic abnormalities in the embryos, markedly increased mCherry expression. In addition, the reporter embryos responded to other POPs, and primary liver cell culture of zebrafish revealed that Cyp1a protein was mainly expressed in the cytoplasm of liver cells. Furthermore, our transgenic fish embryos demonstrated that TCDD exposure can regulate the expression levels of several tumor-related factors, including epidermal growth factor, TNF-α, C-myc, proliferating cell nuclear antigen, TGF-β, serine/threonine kinase (Akt), and phosphorylated Akt, suggesting that our transgenic fish can be used as a sensitive model to evaluate the carcinogenicity induced by TCDD exposure.-Luo, J.-J., Su, D.-S., Xie, S.-L., Liu, Y., Liu, P., Yang, X.-J., Pei D.-S. Hypersensitive assessment of aryl hydrocarbon receptor transcriptional activity using a novel truncated cyp1a promoter in zebrafish.

A Primary Human Liver Cell Culture Model for Hemorrhagic Fever Viruses.

Viral hemorrhagic fevers affect liver functions such as important metabolic processes and the replacement of new blood cells, coagulation factors, and growth factors. Typically, multi-organ diseases such as viral hemorrhagic fevers are studied in an organism, but it is also possible to derive information about the molecular events involved in disease processes by focusing on liver cell culture. Here we describe a multi-cell culture system that is capable of replicating the arenavirus LCMV-WE, a virus that can cause hemorrhagic fever in primates, as a model for liver infection by a hemorrhagic fever virus.

Using a liver cell culture from Epinephelus coioides as a model to evaluate the nonylphenol-induced oxidative stress

The present study aimed to use primary liver cell culture derived from the orange-spotted grouper, Epinephelus coioides, to assess the toxic effects of nonylphenol (NP) on the hepatocyte viability and the liver antioxidant system. E. coioides was selected due to its commercial importance. NP was used in this study because of its high potential of producing oxidative stress due to increased reactive oxygen species (ROS). A liver of E. coioides was digested with PBS containing 0.1% collagenase IV. The digested cells were moved to Leibovitz L-15 culture medium with 20% fetal bovine serum (FBS), 100IUmL−1 penicillin, 100μgmL−1 streptomycin. Aliquots of cell suspension were seeded as a monolayer into sterile 25cm2 tissue culture flasks and incubated at 30°C for 14days. The medium, containing non-attached cells, was removed after 24 to 48h and a new medium was added. The IC50 of 10−4molL−1 was determined for nonylphenol using MTT assay. Cells were then incubated with L-15 medium containing 10−5, 2×10−5, 3×10−5molL−1 of NP and samples were taken after 6, 12 and 24h of incubation for analysis of LPO, SOD, CAT, GPx, LDH, AST, ALT, and ALP. Based on the results, the lowest concentration of NP was not markedly cytotoxic to primary hepatocytes and the cell sensitivity to NP increased dose-dependently. The activities of SOD, CAT and GPx decreased significantly, while activities of LPO, LDH, AST, ALT and ALP, increased significantly in a dose-related pattern in NP-treated cells. In conclusion, this study revealed that NP could induce the oxidative stress in cultivated hepatocytes of E. coioides during a short-term exposure. NP toxicity is mainly due to the induction of the reactive oxygen species (ROS), which lead to cell membrane disruption, damage of cellular metabolism, and interference with cellular macromolecules.

Two-color fluorescent cytosine extension assay for the determination of global DNA methylation.

Here, we present a DNA restriction enzyme-based, fluorescent cytosine extension assay (CEA) to improve normalization and technical variation among sample-to-sample measurements. The assay includes end-labeling of parallel methylation-sensitive and methylation-insensitive DNA restriction enzyme digests along with co-purification and subsequent co-measurement of incorporated fluorescence. This non-radioactive, two-color fluorescent CEA (TCF-CEA) was shown to be a relatively rapid and accurate, with 3-fold greater precision than the one-color CEA. In addition, TCF-CEA provided an index of global DNA methylation that was sensitive to differences >5%. TCF-CEA results were highly correlated with LUminometric Methylation Assay (LUMA) results using human liver cell lines (HepG2, HepaRG, HC-04) as well as a human liver primary cell culture. Hypomethylation was observed in cells treated with the de-methylating agent 5-aza-2'-deoxycytidine. These results demonstrate that TCF-CEA provides a simple method for measuring relative degrees of global DNA methylation that could potentially be scaled up to higher-throughput formats.

Establishment of SV40 large T antigen-immortalized bovine liver sinusoidal cell lines and their immunological responses to deoxynivalenol and lipopolysaccharide.

Immortalized bovine sinusoidal cell lines provide useful tools to study the immunological responses in the liver to the gastrointestinal tract-derived toxic substances, which may cause systemic symptoms in the affected livestock. Here, we established two immortalized bovine liver sinusoidal cell lines, endothelial-like B46, and myofibroblast-like A26, from primary cultures of bovine liver cells by the transfection with SV40 large T antigen. The pro-inflammatory cytokine responses in these cell lines to deoxynivalenol (DON) and lipopolysaccharide (LPS) were then compared to those in the primary bovine Kupffer cells (BKC). BKC were highly responsive to LPS, showing increased levels of IL-1α, IL-1β, IL-6, and TNF-α mRNA 3 h after stimulation. DON induced similar pro-inflammatory cytokine responses in BKC, except for IL-6. The endothelial B46 cells exhibited upregulation of IL-1α, IL-1β, and IL-6 3 h after stimulation by LPS. In contrast to the stimulation by LPS, B46 had relatively low pro-inflammatory cytokine responses to DON, except for IL-1α, which was moderately induced at 3 h and increased at 24 h after stimulation. The myofibroblast-like A26 cells exhibited low responses in the induction of pro-inflammatory cytokines to LPS or DON; however, the expression of IL-6 was significantly observed 3 h after DON stimulation. Our results suggest that bovine liver sinusoidal cells have distinctive pro-inflammatory cytokine responses against harmful substances, and these immune responses might determine the consequence of systemic inflammations in the diseased animal.

Chemical characterization and bioactive properties of Geranium molle L.: from the plant to the most active extract and its phytochemicals.

After a period of indifference, in which synthetic compounds were favored, there is an increasing interest in the study of the biological properties of plants and the active principles responsible for their therapeutic properties. Geranium molle L. has been used in the Portuguese folk medicine for the treatment of various ailments including cancer but, unlike many of the species from the Geranium genus, its phytochemical characterization and biological activity are virtually unexplored. In this study a G. molle sample from Trás-os-Montes, north-eastern Portugal, was chemically characterized regarding nutritional value, free sugars, organic acids, fatty acids and tocopherols, and several aqueous (decoction, infusion) and organic (n-hexane, dichloromethane, ethyl acetate, acetone, methanol) extracts of the plant were assessed for their bioactive properties. The antioxidant activity was evaluated by means of the free radicals scavenging activity, reducing power and inhibition of lipid peroxidation. The cytotoxicity of the different extracts was assessed in vitro against several human cancer cell lines (breast, lung, cervical and hepatocellular carcinomas) and, additionally, their hepatotoxicity was evaluated using a porcine liver primary cell culture. G. molle was shown to be rich in carbohydrates and proteins, providing tocopherols and essential fatty acids. Amongst the various extracts, the acetone extract was found to have the highest content of phenolic compounds (mainly ellagitannins, but also some flavone and flavonol glycosides) as well as the highest antioxidant and cytotoxic activities. To the best of our knowledge, this is the first report on the chemical composition and bioactive properties of G. molle.

Liver sinusoid on a chip: Long-term layered co-culture of primary rat hepatocytes and endothelial cells in microfluidic platforms.

We describe the generation of microfluidic platforms for the co-culture of primary hepatocytes and endothelial cells; these platforms mimic the architecture of a liver sinusoid. This paper describes a progressional study of creating such a liver sinusoid on a chip system. Primary rat hepatocytes (PRHs) were co-cultured with primary or established endothelial cells in layers in single and dual microchannel configurations with or without continuous perfusion. Cell viability and maintenance of hepatocyte functions were monitored and compared for diverse experimental conditions. When primary rat hepatocytes were co-cultured with immortalized bovine aortic endothelial cells (BAECs) in a dual microchannel with continuous perfusion, hepatocytes maintained their normal morphology and continued to produce urea for at least 30 days. In order to demonstrate the utility of our microfluidic liver sinusoid platform, we also performed an analysis of viral replication for the hepatotropic hepatitis B virus (HBV). HBV replication, as measured by the presence of cell-secreted HBV DNA, was successfully detected. We believe that our liver model closely mimics the in vivo liver sinusoid and supports long-term primary liver cell culture. This liver model could be extended to diverse liver biology studies and liver-related disease research such as drug induced liver toxicology, cancer research, and analysis of pathological effects and replication strategies of various hepatotropic infectious agents. .

Counteraction of Apoptotic and Inflammatory Effects of Adriamycin in the Liver Cell Culture by Clinopitolite.

Growing evidence has been reported on adriamycin (ADR) hepatotoxicity in literature. Hepatotoxicity caused by the use of drugs has a serious undesirable effect in the cure of cancer patients that needs to be eliminated. The exact mechanism of ADR on non-cancerous tissue still remains to be a mystery. The zeolite (clinoptilolite) minerals form a complex group of aluminosilicates that often occur as accessory minerals in intermediate and basic rocks. In light of this information, we investigated the possible anti-inflammatory and anti-apoptotic effects of clinoptilolite in ADR that is inducing the toxicity in primary liver cell culture. Primary liver cell culture from rat was used in the study. We had three experiment groups including the following: (1) cells treated only with 50 μM ADR for 24 h, (2) cells treated with the 50 μM ADR for 24 h and then treated with 10(-4) M zeolite for 1 h, and (3) cells were incubated with 50 μM ADR for 24 h and then incubated with 10(-4) M zeolite for 24 h to test its long-term effects. After that, western blotting was performed in order to evaluate protein expression levels of several inflammation markers including IL-1β, tumor necrosis factor (TNF)-α, and nuclear factor kappa B (NF-κB), and immunohistochemistry was carried out to detect apoptosis in liver cell culture. Also, TdT-dUTP Terminal Nick-End Labeling (TUNEL) method was used for detecting apoptosis. We found elevated levels of inflammatory protein and apoptotic markers in ADR-administered cells (p < 0.05). Inflammatory and apoptotic markers decreased significantly after treated with zeolite (p < 0.05). The present study was pointed out that ADR causes hepatotoxicity via apoptosis and/or inflammation processes resulting from initiator NF-κB and TNF which causes proinflammatory mediators such as IL-1β. Elevation of inflammation might give rise to trigger apoptosis. Clinoptilolite counteracted the apoptosis and inflammation induced by ADR arising from the decrease in NF-κB, TNF-α, and IL-1β protein levels.

Role of stress proteins and hormones in bioregulation of ontogeny

In this short review article we tried to overview diffusely spread data on the role of stress proteins and hormones in ontogeny. The work presented here is a product of our long-term studies beginning from the middle of eighties of the last century and performed both in Russia and Brazil. It involves the results obtained with the use of experimental models on laboratory animals in vivo and in vitro, as well as later theoretical research in world literature databases. In experimental studies we used laboratory rats of different age groups and primary cultures of pituitary and liver cells for evaluating respectively body and organ growth and production of immunoreactive growth hormone (GH) and serum albumin (SA), as well as biosynthesis of DNA, total RNA and protein. The results obtained, showing important role of glucocorticoids (GC) in regulation of perinatal pituitary and liver functions and postnatal growth, were reinterpreted by us recently in the frame of DOHaD concept and as related to perinatal imprinting/programming phenomena. It is concluded that the present moment is quite appropriate for the widening of our studies both to the side of early embryonal development and in direction to aging, thus completing the whole cycle of life history / course research, as referred to stress proteins and hormones.

Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation costs.