Dataset Information

In vitro Liver Zonation of Primary Rat Hepatocytes.

ABSTRACT: The ability of the liver to simultaneously carry out multiple functions is dependent on the metabolic heterogeneity of hepatocytes spatially located within a liver lobule spanning from the portal triad to the central vein. This complex zonal architecture of the liver, however, makes accurate in vitro modeling a challenge and often standard culture systems assume a homogenous model which may lead to inaccurate translatability of results. Here, we use a combination of mathematical modeling and experimental data to demonstrate a readily constructible in vitro flow system capable of liver zonation in primary rat hepatocytes. We show the differential expression of zonation markers, enhanced functionality when compared to standard static cultures and zone-specific metabolism and cell damage in the presence of paracetamol, a known zone-specific toxin. This type of advanced system provides a more in-depth and essential understanding of liver physiology and pathophysiology as well as the accurate evaluation of pharmacological interventions at a zone-specific level.

SUBMITTER: Tomlinson L

PROVIDER: S-EPMC6387900 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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<i>In vitro</i> Liver Zonation of Primary Rat Hepatocytes.

Tomlinson Lauren L Hyndman Lauren L Firman James W JW Bentley Robert R Kyffin Jonathan A JA Webb Steven D SD McGinty Sean S Sharma Parveen P

Frontiers in bioengineering and biotechnology 20190218

The ability of the liver to simultaneously carry out multiple functions is dependent on the metabolic heterogeneity of hepatocytes spatially located within a liver lobule spanning from the portal triad to the central vein. This complex zonal architecture of the liver, however, makes accurate <i>in vitro</i> modeling a challenge and often standard culture systems assume a homogenous model which may lead to inaccurate translatability of results. Here, we use a combination of mathematical modeling ...[more]

PMID: 30834246

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Project description:AIM:To investigate the effects of electroporation on primary rat hepatocyte and to optimize the electroporation conditions introducing foreign genes into primary hepatocytes. METHODS:A single-pulse procedure was performed at low voltage (220-400 V) but with high capacitance (500-950 microF). Hepatocytes were divided into 4 groups according to the electroporation conditions: group I, 220 V and 500 microF; group II, 220 V and 950 microF; group III, 400 V and 950 microF,and group IV. The control group was freshly isolated hepatocytes and directly cultured under the same conditions as those of electroporation groups. The effects of electroporation on primary rat hepatocytes were detected by trypan blue exclusion (TBE) and MTT analysis. Besides, albumin (Alb), alanine transaminase (ALT) and lactate dehydrogenase (LDH) in the supernatants of cultured hepatocytes were measured by biochemical assay. RESULTS:Between day 1 and day 15 after incubation, primary rat hepatocytes of each electroporation group appeared normal, being the same with those of control group. TBE staining showed that slight hepatocyte damage and high survival rate were found in the electroporation groups and the control group. Cultured for 3, 7, 11 and 15 days, hepatocyte viability was approximately 92.6+/-2.5 %, 89.5+/-3.3 %, 82.0+/-3.5 % and 74.3+/-1.2 %, respectively. MTT analysis indicated that the viabilities of hepatocytes had no significant difference between each electroporation group, and those were similar to that of control group. At the 36th hour after electroporation, Alb, ALT and LDH in the supernatants of control group were 5.3+/-0.1 g x L(-1), 183.7+/-8.4 nkat x L(-1) and 896.8+/-58.5 nkat x L(-1); those of group II were 5.7+/-0.1 g x L(-1), 215.4+/-16.7 nkat x L(-1) and 1063.8+/-51.8 nkat x L(-1); and those of group III were 5.8+/-0.2 g x L(-1), 217.1+/-8.4 nkat x L(-1) and 1063.8+/-10.0 nkat x L(-1). Statistically, the proteins of group II and group III were significantly higher than those of control group (P<0.05), whereas the protein production of group I, Alb, ALT and LDH were 5.3+/-0.2 g x L(-1), 205.4+/-3.3 nkat x L(-1) and 1035.4+/-116.9 nkat x L(-1), were similar to those of control group. At the same time, TBE and MTT analysis indicated that there was no significant cell viability difference between electroporation groups and control group. CONCLUSION:This single-pulse electroporation procedure performed at low voltage (220-400 V) but with high capacitance (950 microF) is one of the optimal choices to introduce foreign genes into primary rat hepatocyte.

Project description:UnlabelledAngiotensin II (AT-II) is a pro-fibrotic compound that acts via membrane-bound receptors (AT-1R/AT-2R) and thereby activates hepatic stellate cells (HSCs). AT-II receptor blockers (ARBs) are thus important candidates in the treatment of liver fibrosis. However, multiple case reports suggest that AT-1R blockers may induce hepatocyte injury. Therefore, we investigated the effect of AT-II and its receptor blockers on cytokine-, oxidative stress- and bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to TNF-?/Actinomycin D, the ROS-generating agent menadione or the bile salts: glycochenodeoxycholic acid (GCDCA) and tauro-lithocholic acid-3 sulfate (TLCS), to induce apoptosis. AT-II (100 nmol/L) was added 10 minutes prior to the cell death-inducing agent. AT-1R antagonists (Sartans) and the AT-2R antagonist PD123319 were used at 1 µmol/L. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (Sytox green staining) were quantified. Expression of CHOP (marker for ER stress) and AT-II receptor mRNAs were quantified by Q-PCR. AT-II dose-dependently reduced GCDCA-induced apoptosis of hepatocytes (-50%, p<0.05) without inducing necrosis. In addition, AT-II reduced TLCS-induced apoptosis of hepatocytes (-50%, p<0.05). However, AT-II did not suppress TNF/Act-D and menadione-induced apoptosis. Only the AT-1R antagonists abolished the protective effect of AT-II against GCDCA-induced apoptosis. AT-II increased phosphorylation of ERK and a significant reversal of the protective effect of AT-II was observed when signaling kinases, including ERK, were inhibited. Moreover, AT-II prevented the GCDCA-induced expression of CHOP (the marker of the ER-mediated apoptosis).ConclusionAngiotensin II protects hepatocytes from bile salt-induced apoptosis through a combined activation of PI3-kinase, MAPKs, PKC pathways and inhibition of bile salt-induced ER stress. Our results suggest a mechanism for the observed hepatocyte-toxicity of Sartans (angiotensin receptor blockers, ARBs) in some patients with chronic liver injury.

Dataset Information

In vitro Liver Zonation of Primary Rat Hepatocytes.

Publications

<i>In vitro</i> Liver Zonation of Primary Rat Hepatocytes.

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