Porcine Hepatocytes Research Articles

Preparation and Characterization of Alginate-Chitosan Microcapsule for Hepatocyte Culture.

This method is used to prepare microencapsulation of hepatocytes. The key steps in the protocol are (a) isolation of porcine hepatocytes, followed by (b) production of hepatocytes encapsulation, and (c) evaluation of encapsulations and hepatocytes. This method of collecting porcine hepatocytes was first used in 2003 (Maruyama et al. Cell Transplant 12:593-598, 2003). Cell encapsulation technology was first introduced by Chang (Science 146:524-525, 1964) and later by Lim and Sun in 1980 (Science 210:908-910, 1980). Since then, it has been well adopted in tissue engineering and controlled delivery of biological therapeutics such as bioartificial liver.

Comparison of methods for isolating primary hepatocytes from mini pigs

Successful porcine hepatocyte isolation is crucial for the development of bioartificial liver devices and hepatocyte transplantation. Serva collagenase NB grades are formulated collagenases that are suitable for various tissue isolation applications. N-acetylcysteine (NAC) can improve the viability of human hepatocytes. The aim of this study was to compare the effectiveness of two collagenases and effect of NAC on hepatocyte isolation from porcine liver tissue. Porcine hepatocytes were isolated using the perfusion method from Bama mini pigs assigned to the Serva NB 4 group (n=6), the Serva NB 8 group (n=6), or the NB 8+NAC group (n=6). Viability and yield were defined as fresh hepatocytes and their spheroids formation after 24-hour rocker culture in serum-free medium. Metabolic function was assessed by gene expression, albumin, and urea synthesis. All procedures resulted in successful hepatocyte isolation. Cells from the NB 8+NAC group had (97.8±1.9)% viability, which was higher than the NB 8 group with (94.4±2.4)% and the NB 4 group with (94.5±3.2)% (P<.001). The final cell yield reached (11.8±1.0)×10(9) cells in the NB 8+NAC group, compared to (9.5±2.1)×10(9) cells in the NB 8 group (P<.01) and (9.1±1.1) ×10(9) cells in the NB 4 group (P<.001). The secretion of albumin was superior in the NB 8+NAC group at a concentration of (425.8±35.3) ng/mL compared to the NB 8 group (339.1±32.6) ng/mL (P <.001) and NB 4 group (293.6±43.3) ng/mL (P <.01). The injury of hepatocytes also decreased in the NB 8+NAC group (P<.01). The data are presented as means ± SD. Formulated collagenase Serva NB 8 and NAC could improve the porcine hepatocyte isolation, resulting in higher yields of viable cells.

The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway

It has been implicated that IGF-1 secretion can be regulated by dietary protein. However, whether the dipeptides, one of digested products of dietary protein, have influence on IGF-1 secretion remain largely unknown. Our study aimed to investigate the effects of the dipeptide Pro-Asp on IGF-1 secretion and expression in hepatocytes and to explore the possible underlying mechanisms. Our findings demonstrated that Pro-Asp promoted the secretion and gene expression of IGF-1 in HepG2 cells and primary porcine hepatocytes. Meanwhile, Pro-Asp activated the ERK and Akt signaling pathways, downstream of IGF-1. In addition, Pro-Asp enhanced GH-mediated JAK2/STAT5 signaling pathway, while inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Asp on IGF-1 secretion and expression. Moreover, acute injection of Pro-Asp stimulated IGF-1 expression and activated JAK2/STAT5 signaling pathway in mice liver. Together, these results suggested that the dipeptide Pro-Asp promoted IGF-1 secretion and expression in hepatocytes by enhancing GH-mediated JAK2/STAT5 signaling pathway.

Abstract 2050: Porcine drug metabolism and toxicity in vitro model utilizing transformed hepatocyte cell lines (HepCre)

Abstract To date, in vitro cytotoxicity assays are not highly predictive of in vivo toxicity. The adverse effects of new drugs are often not discovered until preclinical animal safety studies or even clinical trials; 40% of drugs drop out in preclinical animal studies and 89% of those that reach clinical trials fail. There is a critical need for more predictive and reliable in vitro testing methods. Due to its physiological similarities with humans, pigs have emerged as a suitable and reliable animal model for pharmacological and toxicological studies. To further the pigs’ suitability, we have developed and characterized a transformed porcine hepatocyte cell line to support drug toxicity and metabolism assessments. Primary porcine hepatocytes isolated by a modified procedure of Panda's method (Panda et al., 2015) express phase I and II drug metabolism, phase-III transport, and nuclear receptors involved in regulation of drug metabolism transcripts. However, primary hepatocytes have a limited life span in culture and usually within 8 days post culture more than 50% of cells undergo apoptosis. Moreover, normal gene expression declines from day 5 in culture. To overcome these limitations, we have generated and characterized transformed hepatocyte cell lines (HepCre) derived from the transgenic Oncopig. Hepatocytes were isolated from Oncopigs (Schook et at., 2015) and transformed by treatment with Cre recombinase. When cultured in the presence of a differentiating agent (DMSO), the HepCre cell lines maintained normal hepatic functions, as well as the potential to express all the main drug metabolism and regulation genes comparable to that of cultured primary hepatocytes. The effect of model CYP (cytochrome P450) inducers (3- methylcholanthrene, rifampicin and phenobarbital) on the expression of CYP enzymes in primary hepatocytes and HepCre were studied. Treatment of 3-methylcholanthrene caused a significant upregulation of CYPA1 and CYPA2 transcripts, but the transcript of other CYPs remain unchanged. Both rifampicin and phenobarbital exposure resulted in the upregulation of several CYP transcripts, including CYP2A19, CYP2B22, and CYP3A. The gene transcription patterns in HepCre were similar to those of primary hepatocytes and in vitro human models. Toxicity responses of HepCre cells to hepatotoxic drugs (i.e. aflatoxin B1, chlorpromazine, amiodarone, and acetaminophen) was evaluated. After 72 hr of exposure, cell viability decreased in a concentration-dependent manner with IC50 of 5 μm, 50 μm, 20 μm, and 70 μm for aflatoxin B1, amiodarone, chlorpromazine, and acetaminophen, respectively. These findings indicate that this porcine transformed HepCre cell line represents a useful and predictive model for high throughput screening of new drugs as well as studies on metabolism and hepatotoxicity of chemicals.

Efficacy of Fluidized Bed Bioartificial Liver in Treating Fulminant Hepatic Failure in Pigs: A Metabolomics Study.

Bioartificial livers may act as a promising therapy for fulminant hepatic failure (FHF) with better accessibility and less injury compared to orthotopic liver transplantation. This study aims to evaluate the efficacy and safety of a fluidized bed bioartificial liver (FBBAL) and to explore its therapeutic mechanisms based on metabolomics. FHF was induced by D-galactosamine. Eighteen hours later, pigs were treated with an FBBAL containing encapsulated primary porcine hepatocytes (B group), with a sham FBBAL (containing cell-free capsules, S group) or with only intensive care (C group) for 6 h. Serum samples were assayed using ultra-performance liquid chromatography-mass spectrometry. The difference in survival time (51.6 ± 7.9 h vs. 49.3 ± 6.6 h) and serum metabolome was negligible between the S and C groups, whereas FBBAL treatment significantly prolonged survival time (70.4 ± 11.5h, P < 0.01) and perturbed the serum metabolome, resulting in a marked decrease in phosphatidylcholines, lysophosphatidylcholines, sphingomyelinase, and fatty acids and an increase in conjugated bile acids. The FBBAL exhibits some liver functions and may exert its therapeutic effect by altering the serum metabolome of FHF pigs. Moreover, alginate–chitosan capsules have less influence on serum metabolites. Nevertheless, the alterations were not universally beneficial, revealing that much should be done to improve the FBBAL.

Preparation and Characterization of an Antibody Antagonist That Targets the Porcine Growth Hormone Receptor.

A series of antagonists specifically targeting growth hormone receptors (GHR) in different species, such as humans, rats, bovines, and mice, have been designed; however, there are currently no antagonists that target the porcine growth hormone (GH). Therefore, in this study, we developed and characterized a porcine GHR (pGHR) antibody antagonist (denoted by AN98) via the hybridoma technique. The results from enzyme-linked immunosorbent assay, fluorescence activated cell sorter, indirect immunoinfluscent assay, and competitive receptor binding analysis showed that AN98 could specifically recognize pGHR, and further experiments indicated that AN98 could effectively inhibit pGH-induced signalling in CHO-pGHR cells and porcine hepatocytes. In addition, AN98 also inhibited GH-induced insulin-like growth factor-1 (IGF-1) secretion in porcine hepatocytes. In summary, these findings indicated that AN98, as a pGHR-specific antagonist, has potential applications in pGH-pGHR-related research on domestic pigs.

Use of a Closed Culture System to Improve the Safety of Lentiviral Vector Production.

We evaluated the possibility of introducing a combination of six oncogenes into primary porcine hepatocytes (PPH) using a lentiviral vector (LV)-mediated gene transfer in order to develop a porcine hepatocellular carcinoma model based on autologous transplantation of ex vivo-transformed hepatocytes. The six oncogenes were introduced into three plasmids, hence enabling the production of LVs encoding a luciferase reporter gene and hTERT+p53(DD), cyclinD1+CDK4(R24C), and c-myc(T58A)+HRas(G21V) genes, respectively. In order to improve the protection of the laboratory personnel manipulating such LVs, we used a compact cell culture cassette (CliniCell(®) device) as a closed cell culture system. We demonstrated that the CliniCell device allows to produce LVs, through plasmid transfection of 293T cells, and, after transfer to a second cassette, to transduce PPH with a similar efficacy as conventional open cell culture systems such as flasks or Petri dishes. Additionally, it is possible to cryopreserve at -80°C the transduced cells, directly in the CliniCell device used for the transduction. In conclusion, the use of a closed culture system for the safe handling of oncogene-encoding LVs lays the foundation for the development of porcine tumor models based on the autologous transplantation of ex vivo-transformed primary cells.

Confocal Imaging and Tissue-Specific Fluorescent Probes for Real-Time In Vivo Immunohistochemistry. Proof of the Concept in a Gastric Lymph Node Metastasis Model.

Tumor-specific fluorescent antibodies, which can be recognized at a cellular or tissue level using optical imaging such as confocal laser endomicroscopy (CLE), could provide a means for rapid and accurate tumor diagnosis and staging. The aim of this study was to evaluate the ability of CLE to detect the presence of tagged cells within lymph nodes in an original simulated metastatic model. A solution of indocyanine green containing a suspension of porcine hepatocytes, marked with carboxy-fluorescein-succinimidyl-ester (CFSE), was injected endoscopically in the gastric submucosa of 10 pigs. Fluorescence lymphography using a near-infrared laparoscope was used to identify sentinel and secondary drainage nodes. Additionally, a nonfluorescent gastric and a mesenteric node were identified. Every 5-10min, those nodes were scanned using probe-based or needle-based CLE (pCLE or nCLE). Immunohistochemistry (IHC) using anti-cytokeratin 18 antibodies was subsequently performed to confirm the presence of hepatocytes in the lymph nodes. A total of 36 lymph nodes were analyzed with both CLE probes. Hepatocyte penetration in lymph nodes, as assessed by repeated CLE scanning, took 10-40min after submucosal injection. Concordance between CLE and IHC was 84 and 72% for pCLE and nCLE, respectively. False negatives were partly due to incomplete CFSE labeling of hepatocytes, which could not be recognized by CLE, but were detected with IHC. Real-time CLE analysis effectively recognized the presence in perigastric nodes of marked hepatic cells that had been injected endoscopically in the stomach. Validation studies on tumor-bearing animals using tumor-specific antibodies should be performed.

No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes

A novel hybrid bioartificial liver (HBAL) was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs). This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses (PERVs) into canines with acute liver failure (ALF) undergoing HBAL. Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity. PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative. No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.

Pivotal preclinical trial of the spheroid reservoir bioartificial liver

The neuroprotective effect of the spheroid reservoir bioartificial liver (SRBAL) was evaluated in a porcine model of drug-overdose acute liver failure (ALF). Healthy pigs were randomized into three groups (standard therapy (ST) alone, ST+No-cell device, ST+SRBAL device) before placement of an implantable intracranial pressure (ICP) monitor and a tunneled central venous catheter. One week later, pigs received bolus infusion of the hepatotoxin D-galactosamine and were followed for up to 90h. At 48h, all animals had developed encephalopathy and biochemical changes confirming ALF; extracorporeal treatment was initiated and pigs were observed up to 90h after drug infusion. Pigs treated with the SRBAL, loaded with porcine hepatocyte spheroids, had improved survival (83%, n=6) compared to ST alone (0%, n=6, p=0.003) and No-cell device therapy (17%, n=6, p=0.02). Ammonia detoxification, peak levels of serum ammonia and peak ICP, and pig survival were influenced by hepatocyte cell dose, membrane pore size and duration of SRBAL treatment. Hepatocyte spheroids remained highly functional with no decline in mean oxygen consumption from initiation to completion of treatment. The SRBAL improved survival in an allogeneic model of drug-overdose ALF. Survival correlated with ammonia detoxification and ICP lowering indicating that hepatocyte spheroids prevented the cerebral manifestations of ALF (brain swelling, herniation, death). Further investigation of SRBAL therapy in a clinical setting is warranted.

Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture

Alpha-1 acid glycoprotein (AGP, orosomucoid, ORM-1) is a highly glycosylated mammalian acute-phase protein, which is synthesized primarily in the liver and represents the major serum protein in newborn pigs. Recent data have suggested that the pig is unique in that AGP is a negative acute-phase protein in this species, and its circulating concentration appears to be associated with growth rate. The purpose of the present study was to investigate the regulation of AGP synthesis in hepatocytes prepared from suckling piglets and to provide a framework to compare its regulation with that of haptoglobin (HP), a positive acute-phase protein. Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h. The influences of hormones, cytokines, and redox modifiers on the expression and secretion of AGP and HP were determined by relative polymerase chain reaction and by measuring the concentration of each protein secreted into culture medium. The messenger RNA abundance and/or secretion of AGP protein was enhanced by interleukin (IL)-17a, IL-1, and resveratrol and inhibited by tumor necrosis factor-α (TNF), oncostatin M, and thyroid hormone (P < 0.05). HP expression and synthesis were upregulated by oncostatin M, IL-6, and dexamethasone and downregulated by TNF (P < 0.01). The overall messenger RNA expression at 24 h was in agreement with the secreted protein patterns confirming that control of these proteins in hepatocytes is largely transcriptional. Moreover, these data support the consideration that AGP is a negative acute-phase reactant and appears to be regulated by cytokines (with the exception of TNF) and hormones primarily in a manner opposite to that of the positive acute-phase protein, HP.

Development and Characterization of a Novel Anti-idiotypic Monoclonal Antibody to Growth Hormone, Which Can Mimic Physiological Functions of Growth Hormone in Primary Porcine Hepatocytes.

B-32 is one of a panel of monoclonal anti-idiotypic antibodies to growth hormone (GH) that we developed. To characterize and identify its potential role as a novel growth hormone receptor (GHR) agonist, we determined that B-32 behaved as a typical Ab2β based on a series of enzyme-linked immunosorbent assay assays. The results of fluorescence-activated cell sorting, indirect immunofluorescence and competitive receptor binding assays demonstrated that B-32 specifically binds to the GHR expressed on target cells. Next, we examined the resulting signal transduction pathways triggered by this antibody in primary porcine hepatocytes. We found that B-32 can activate the GHR and Janus kinase (2)/signal transducers and activators of transcription (JAK2/STAT5) signalling pathways. The phosphorylation kinetics of JAK2/STAT5 induced by either GH or B-32 were analysed in dose-response and time course experiments. In addition, B32 could also stimulate porcine hepatocytes to secrete insulin-like growth factors-1. Our work indicates that a monoclonal anti-idiotypic antibody to GH (B-32) can serve as a GHR agonist or GH mimic and has application potential in domestic animal (pig) production.

Deoxidation rates play a critical role in DNA damage mediated by important synthetic drugs, quinoxaline 1,4-dioxides.

Quinoxaline 1,4-dioxides (QdNOs) are synthetic agents with a wide range of biological activities. However, the mechanism of DNA damage mediated by QdNOs is far from clear. Five classical QdNOs, quinocetone (QCT), mequindox (MEQ), carbadox (CBX), olaquindox (OLA), and cyadox (CYA), were used to investigate the genotoxicity of QdNOs. The deoxidation rate of QdNOs was presumed to play a role in their genotoxicity. Deoxidation rates of QdNOs in both rat and pig liver microsomes were investigated using LC/MS-IT/TOF, and their relative quantification was achieved with HPLC. To reveal the relationships between the deoxidation rate and genotoxicity, cell damage, oxidative stress, and DNA damage were detected. Under low oxygen conditions, the rank order of the desoxy and bidesoxy rates in rat and pig liver microsomes was QCT < CBX < MEQ < OLA < CYA and QCT < MEQ < CBX < OLA < CYA, respectively. Only desoxy-quinoxalines were detected under aerobic conditions. The concentrations of deoxidized metabolites under low oxygen conditions were at least 6 times higher than those under aerobic conditions. In rats, porcine primary hepatocytes, and HepG2 cells, oxidative stress indices and DNA damage showed inverse relationships with the deoxidation rate, indicating that the deoxidation rate of QdNOs, especially bidesoxy rates, might play a critical role in mediating their ability to promote DNA damage. These results indicated that faster deoxidation of QdNOs results in lower DNA-damage-induced toxicity. Our results shed new light on the prevention of DNA damage mediated by QdNOs and help to understand the relationships among the chemical structures, metabolism, and DNA damage of QdNOs.

Potential Barriers to Human Hepatocyte Transplantation in MUP–uPAtg(+/+)Rag2-/-γC-/-Mice

Primary human fetal and adult hepatocytes have been considered feasible donor cell sources for cell transplantation. We compared the engraftment efficiencies between adult human, fetal human, and adult porcine hepatocytes after transplantation into MUP-uPA(tg(+/+))Rag2(-/-)γC(-/-)mice. Transplantation of adult human hepatocytes yielded a 1,000-fold higher serum albumin level compared to transplantation of fetal human hepatocytes, while transplantation of adult porcine hepatocytes resulted in a 100-fold higher serum albumin level than adult human hepatocytes. These results suggest that adult liver cells are superior to fetal liver cells for transplantation, and caution should be applied if porcine hepatocytes are used for preclinical studies as a proof of concept for human hepatocytes.

IFN-γ regulates cytochrome 3A29 through pregnane X receptor in pigs

1. The expression and the activity of cytochromes P450 (CYPs) can be elevated by the activation of nuclear receptors. The pregnane X receptor (PXR, or nuclear receptor NR1I2) is a ligand-activated transcription factor that mediates responses to diverse xenobiotics and endogenous chemicals. Here we investigated the regulatory role of PXR in IFN-γ-mediated CYP3A29 expression in pig liver microsomes, primary porcine hepatocytes, and a cultured hepatocyte cell line.2. IFN-γ significantly up-regulated CYP3A29 and PXR expressions at mRNA and protein levels in a dose-dependent manner. IFN-γ treatment significantly increased the metabolism of nifedipine. PXR and IFN-γ treatments significantly enhanced the activity of CYP3A29 promoter and the upstream region from −1473 to −1021 of CYP3A29 might be PXR-binding site. Moreover, the IFN-γ-induced CYP3A29 expression was blocked by PXR knockdown, whereas CYP3A29 mRNA and protein expression levels were dramatically elevated by PXR overexpression.3. The regulatory effect of IFN-γ on CYP3A29 expression is mediated via PXR.

Comparative proteomic analysis of primary hepatocytes co-cultured with bone marrow mesenchymal stem cells in vitro

Objective To identify the differentially expressed proteins in hepatocytes within the optimal co-cultivation system with bone marrow mesenchymal stem cells (MSCS).Methods The optimal co-cultivation of MSCS and primary hepatocytes was generated in vitro.CD44-CD45-cell population (hepatocytes) was sorted from co-culture system by flow cytometry.Protein samples were isolated by twodimensional (2D) gel electrophoresis.Bioinformatics was employed to analyze the relevance between the altered proteins.The protein expression levels of interest were confirmed by Western blotting.Results Thirty-four altered protein spots were unambiguously identified with high confidence,which are likely associated with hepatic metablic functions,cell cycle,cellular proliferation and apoptosis,as well as immune modulation.Conclusion Several differentially expressed functional proteins within co-cultured hepatocytes have been revealed by comparative proteomics,which may be associated with enhanced hepatic functionality,prolonged cellular survival,and immune modulation. Key words: Porcine hepatocytes ; Bone marrow mesenchymal stem cells ; Co-culture ; Proteome

A sensitive method for the analysis of glutathione in porcine hepatocytes

Background. Reduced glutathione (γ-glutamylcysteinylglycine), GSH, is essential when protecting cells from oxidative stress and also an indicator of disease risk. Reported concentrations of GSH and its oxidized form, glutathione disulfide (GSSG), varies considerably, primarily due to the instability of GSH and various analytical methods. Methods. We designed a sensitive method to analyze GSH and GSSG in porcine hepatocytes using liquid chromatography–tandem mass spectrometry (LC–MS/MS) after stabilization with N-ethylmaleimide (NEM). This method includes stable isotope labeled internal standards and simple synthesis of labeled GSSG which commercial sources rarely offer. GSH and GSSG were analyzed in porcine liver biopsies giving a reference interval based on a large number of samples (26 pigs; 3 parallels). Results. The LC-MS/MS results revealed excellent linearity for both GSH and GSSG, with interday coefficient of variation (%CV) for GSH-NEM and GSSG <10 %. Accuracy for recovery tests was between 95.6% and 106.7% (n = 3) for GSH and between 92.3% and 107.7% (n = 3) for GSSG. The limits of quantification were 0.1 μM for GSH-NEM and 0.08 μM for GSSG. The mean concentration of GSH was 3.5 (95% CI = 1.5–8.1) mmol/liter and of GSSG 0.0023 (95% CI = 0.0003–0.019) mmol/liter. Conclusion. For the first time GSH and GSSG are analyzed in porcine hepatocytes by LC-MS/MS yielding a reference level of GSH and GSSG. The method is reproducible in any laboratory with LC-MS/MS service and will probably be applicable in all soft tissues and cell suspensions, essentially with no modification.

A Device for Albumin and Cytosol Detoxication

A new detoxication method based the Bioartificial Liver apparatus and the use of human serum albumin (HSA) and porcine hepatocyte cytosol (PHC) is described. The method is based on the interaction of HSA and PHC with the patient’s blood through a semi-permeable membrane. The apparatus consists of a bioreactor, hemodialyzer, and blood line as well as thermoregulation, oxygenation, and procedure safety systems. The PHC and HSA dialyzing media were tested using the Bioartificial Liver apparatus. They appeared to be highly effective and capable of reducing biochemical markers of cytolysis, cholestasis, and endogenous intoxication in patients without any adverse side effects. Thus, the method can be recommended for further clinical tests and introduction into intensive care practice.

Effects of Lactobacillus plantarum 2142 and sodium n-butyrate in lipopolysaccharide-triggered inflammation: comparison of a porcine intestinal epithelial cell line and primary hepatocyte monocultures with a porcine enterohepatic co-culture system.

This study was based on our previously developed double-layered enterohepatic co-culture system, composed of nontumorigenic porcine intestinal epithelial cell line (IPEC-J2) and primary culture of porcine hepatocytes. The anti-inflammatory effect of spent culture supernatant of Lactobacillus plantarum 2142 (Lp2142; 13.3%) and sodium n-butyrate (2 mM) was tested on IPEC-J2 and hepatocyte monocultures as well as on the gut-liver co-culture. To mimic inflammation, lipopolysaccharide (LPS; 1 and 10 μg/mL) was applied. Production of IL-8 and IL-6 was measured as a marker of inflammatory responses. The paracellular permeability of the intestinal epithelium was also monitored by fluoresceinisothiocyanate-labeled dextran 4 assay. Significant increase of IL-8 concentration was observed in the IPEC-J2 monoculture (P < 0.01) while the level of IL-6 was not changed following LPS treatment. Concentration of IL-8 and IL-6 was grown significantly in hepatocyte monocultures (P < 0.05 and P < 0.001) as well as in the co-culture after 10 μg/mL LPS treatment (P < 0.001 and P < 0.001). One microgram per milliliter LPS caused elevated IL-8 level in the co-culture (P < 0.001) and in the hepatocyte monoculture (P < 0.01), while it caused increased IL-6 level only in the hepatocytes (P < 0.001). Production of IL-8 was significantly decreased by butyrate in case of 1 μg/mL as well as 10 μg/mL LPS exposure in the co-culture (P < 0.001). Application of butyrate also reduced IL-6 level in the co-culture after 10 μg/mL LPS treatment (P < 0.01). Lactobacillus plantarum 2142 decreased IL-8 level after incubation with 1 μg/mL LPS (P < 0.001), while in case of 10 μg/mL LPS treatment only a marginal lowering in IL-8 (P = 0.064) release was measured. The IL-6 concentration was significantly reduced (P < 0.01 in case of 1 μg/mL LPS treatment) by Lp2142 in the co-culture. Contrarily, the elevated IL-8 and IL-6 level of hepatocytes has not been reduced in case of either butyrate or Lp2142 addition. The enterohepatic co-culture model offers a possibility for fast and reliable screening of new candidates against enteric inflammation, which are of special interest in porcine medicine and health management. According to our results, Lp2142 and butyrate both seem to be effective as anti-inflammatory agents in LPS-triggered inflammatory response, tested in the gut-liver co-culture model.

Pregnane X receptor is required for IFN-α-mediated CYP3A29 expression in pigs

Pregnane X receptor (PXR) has been identified as a central mediator for coordinate responses to xenobiotic and drug metabolism, and is the major transcriptional regulator of cytochrome P-450 (CYP). Interferon (IFN)-α is known to induce antiviral mechanisms and exert immune regulatory capacity in various cell types. Here, we used primary porcine hepatocytes and a cultured hepatocyte cell line to identify the metabolic role of PXR in IFN-α-mediated CYP3A29 expression. We found that IFN-α could activate PXR in both time- and dose-dependent manners in pigs. Activation of PXR significantly increased CYP3A29 mRNA and protein expression. Meanwhile, the expression of CYP3A29 induced by IFN-α occurred after the increase of PXR expression in porcine hepatocytes. In addition, the IFN-α-induced CYP3A29 expression was blocked by PXR knockdown. The PXR-overexpressed cells (transfected with porcine PXR) increased CYP3A29 mRNA and protein expression. Furthermore, in animal experiments, we found that IFN-α increased both CYP3A29 mRNA and protein levels. Collectively, our results suggest that PXR plays an important role in IFN-α-mediated CYP3A29 expression in porcine hepatocytes.