Proliferation Of Rat Hepatocytes Research Articles

Metabolic reprogramming in Nrf2-driven proliferation of normal rat hepatocytes.

Cancer cells reprogram their metabolic pathways to support bioenergetic and biosynthetic needs and to maintain their redox balance. In several human tumors, the Keap1-Nrf2 system controls proliferation and metabolic reprogramming by regulating the pentose phosphate pathway (PPP). However, whether this metabolic reprogramming also occurs in normal proliferating cells is unclear. To define the metabolic phenotype in normal proliferating hepatocytes, we induced cell proliferation in the liver by 3 distinct stimuli: liver regeneration by partial hepatectomy and hepatic hyperplasia induced by 2 direct mitogens: lead nitrate (LN) or triiodothyronine. Following LN treatment, well-established features of cancer metabolic reprogramming, including enhanced glycolysis, oxidative PPP, nucleic acid synthesis, NAD + /NADH synthesis, and altered amino acid content, as well as downregulated oxidative phosphorylation, occurred in normal proliferating hepatocytes displaying Nrf2 activation. Genetic deletion of Nrf2 blunted LN-induced PPP activation and suppressed hepatocyte proliferation. Moreover, Nrf2 activation and following metabolic reprogramming did not occur when hepatocyte proliferation was induced by partial hepatectomy or triiodothyronine. Many metabolic changes in cancer cells are shared by proliferating normal hepatocytes in response to a hostile environment. Nrf2 activation is essential for bridging metabolic changes with crucial components of cancer metabolic reprogramming, including the activation of oxidative PPP. Our study demonstrates that matured hepatocytes exposed to LN undergo cancer-like metabolic reprogramming and offers a rapid and useful in vivo model to study the molecular alterations underpinning the differences/similarities of metabolic changes in normal and neoplastic hepatocytes.

In vitro proliferation and long-term preservation of functional primary rat hepatocytes in cell fibers

Primary hepatocytes are essential cellular resources for drug screening and medical transplantation. While culture systems have already succeeded in reconstituting the biomimetic microenvironment of primary hepatocytes, acquiring additional capabilities to handle them easily as well as to expand them remains unmet needs. This paper describes a culture system for primary rat hepatocytes, based on cell fiber technology, that brings scalability and handleability. Cell fibers are cell-laden core–shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium via the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated their proliferation while maintaining their viability and their hepatic specific functions for up to thirty days of subsequent culture. We then demonstrated the efficiency of proliferating primary rat hepatocytes in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Our culture system could therefore be included in innovative strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.

Reciprocal regulation of glycogen synthase kinase-3 and β-catenin affects cell proliferation of fructose treated rat hepatocytes

High consumption of fructose might lead to obesity, diabetes, and metabolic syndrome in the long term. Recent studies demonstrated the induction of insulin resistance in the liver tissues by down-regulation of insulin signaling pathway elements. Glycogen synthase kinase-3 (GSK-3), one of the insulin signaling elements, suppresses the β-catenin function that is required for cell proliferation and integrity. This study is designed to demonstrate the effects of fructose on the proliferation of rat hepatocytes and its effects on GSK-3 and β-catenin expression. Accordingly, rat hepatocytes were treated with different concentrations of fructose, and cell proliferation was followed with an xCELLigence real-time cell analysis system. Besides, gene and protein expression levels of GSK3β and β-catenin were evaluated in fructose-treated cells with qRT-PCR and Western blot, respectively. The results demonstrated proliferative effects of fructose at low doses (0-25-50 mM), but cytotoxic properties are pronounced at higher doses (100-150 mM). The IC50 value was calculated as 140±7 mM fructose for Clone-9 cells. Molecular effects of fructose over GSK-3β and β-catenin appeared at gene and protein levels at 100- and 150-mM concentrations at which GSK-3β were suppressed. Conversely, high-dose fructose leads to β-catenin induction as a compensatory mechanism to counteract the antiproliferative effects of fructose at these doses. In conclusion, high-dose fructoseinduced cytotoxicity activates a compensatory molecular mechanism involving β-catenin induction which might protect the cells in the long-term fructose exposure

Formyl Peptide Receptor 2 Alleviates Hepatic Fibrosis in Liver Cirrhosis by Vascular Remodeling.

Hexapeptide WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met), a ligand of formyl peptide receptor 2, exhibits anti-inflammatory and angiogenic properties in disease models. However, the therapeutic effects of WKYMVm on hepatic fibrosis have not been evaluated to date. Therefore, we investigated whether WKYMVm exerts antifibrotic effects and induces vascular regeneration in a rat model of bile duct ligation (BDL). The antifibrotic and angiogenic effects of WKYMVm on liver regeneration in the BDL rat model were analyzed using biochemical assays, qRT-PCR, western blotting, immunofluorescence, and immunohistochemistry. To determine the effects of WKYMVm on hepatic fibrosis and angiogenesis in vitro, we measured the expression levels of fibrotic factors in hepatic stellate cells (HSCs) and angiogenic factors in human umbilical vein endothelial cells (HUVECs). WKYMVm attenuated the expression of collagen type I (Col I) and α-smooth muscle actin (α-SMA) and significantly increased the levels of angiogenetic factors in the BDL model (p < 0.05). WKYMVm reduced fibrotic marker expression in transforming growth factor (TGF)-β-induced HSCs and promoted angiogenic activity through tube formation in 5-Fluorouracil (FU)-treated HUVECs (p < 0.05). Also, WKYMVm administration enhanced hepatocyte proliferation in BDL rats (p < 0.05). The WKYMVm alleviates hepatic fibrosis by inhibiting HSC activation and promotes hepatic regeneration via vascular remodeling. These data suggest that the WKYMVm may be a new therapeutic agent for liver fibrosis.

Peroxisome proliferator-activated receptor α agonist-induced histidine decarboxylase gene expression in the rat and mouse liver.

By analysis of the data from the Toxicogenomics Database (TG-GATEs), histidine decarboxylase gene (Hdc) was identified as largely and commonly upregulated by three fibrates, clofibrate, fenofibrate, and WY-14,643, which are known to induce hepatocellular hypertrophy and proliferation via stimulation of peroxisome proliferator-activated receptor α (PPARα) in rodents. As histamine has been reported to be involved in the proliferation of liver cells, the present study was conducted to focus on Hdc. Among other genes related to histidine and histamine, the expression of the gene of histamine ammonia lyase (Hal) was exclusively mobilized by the three fibrates. The expression of Hdc, which was usually very low in the liver, was increased with the repeated administration of fibrates, and concomitantly, the constitutive expression of Hal was suppressed. An interpretation is that the formation of urocanic acid from histidine under the normal condition switches to the formation of histamine. The mobilization of gene expression of Hdc and Hal by PPARα agonists could not be reproduced in primary cultured hepatocytes. The Hdc mRNA appeared to be translated to a protein which is processed differently from brain but similarly to gastric mucosa. Surprisingly, the fibrates caused hepatic hypertrophy but no induction of Hdc mRNA at all in mice. These results revealed that the changes in the histidine catabolism by PPARα agonists might be partially, but not directly, involved in the hepatocyte proliferation in rats, and there is a large genetic distance even between rat and mouse.

MiR-199a-5p regulates rat liver regeneration and hepatocyte proliferation by targeting TNF-α TNFR1/TRADD/CASPASE8/CASPASE3 signalling pathway

Abnormally expressed miR-199a-5p (miR-199a) has been frequently reported in multiple types of malignancies. Nevertheless, its effect in liver regeneration (LR) is largely still unclear. Herein, we investigated the function of miR-199a in hepatocyte proliferation during LR. As a result, miR-199a expression was significantly increased 12–30 h, in rat hepatic tissue, after partial hepatectomy (PH). The down-regulated expression of miR-199a inhibited proliferation as well as promoted cell apoptosis of BRL-3A. Additionally, TNF-α was found as a target of miR-199a. The administration of TNF-α siRNA regulated the effects of miR-199a on hepatocyte proliferation as well as miR-199a-modulated TNF-α/TNFR1/TRADD/CASPASE8/CASPASE3 signalling pathways. Taken together, these present findings suggested that miR-199a promoted hepatocyte proliferation as well as LR via targeting TNF-α/TNFR1/TRADD/CASPASE8/CASPASE3.

Osteopontin promotes rat hepatocyte proliferation both in vitro and in vivo

Aim This study aimed to examine the effects of osteopontin (OPN) on hepatocyte growth and liver regeneration (LR). Methods A recombinant lentivirus expressing OPN and OPN-siRNAs were used to treat BRL-3A cells, while the adenovirus expressing OPN or OPN-targeted shRNA were applied for rat primary hepatocytes. Moreover, rrOPN and OPN-Ab were added to treat BRL-3A. Next, rrOPN was administrated into rat regenerating livers. Then in vitro and in vivo assays were performed to evaluate the biological function of OPN in hepatocyte growth and LR. Results OPN overexpression facilitated proliferation and viability of BRL-3A cells and primary hepatocytes, while OPN silencing reversed these effects. Similarly, rrOPN stimulated cell cycle progression and viability, but OPN-Ab led to cell cycle arrest and decreased viability. OPN overexpression induced the expression of p-STAT3, p-AKT and CCND1, and OPN siRNA led to reduction of p-AKT and CCND1. Furthermore, rrOPN promoted the expression of p-STAT3 and p-AKT, while OPN-Ab and PI3K/Akt inhibitor LY294002 both inhibited the expressions of p-AKT and Bcl2. Moreover, LR rate, serum IL-6 and TNF-α, Ki-67+ proportion and the phosphorylation of STAT3, AKT and p65 were augmented by rrOPN treatment. Conclusion OPN promotes hepatocyte proliferation both in vitro and in vivo through STAT3 and AKT signaling pathways.

Gene selection of rat hepatocyte proliferation using adaptive sparse group lasso with weighted gene co-expression network analysis

Grouped gene selection is the most important task for analyzing the microarray data of rat liver regeneration. Many existing gene selection methods cannot outstand the interactions among the selected genes. In the process of rat liver regeneration, one of the most important events involved in many biological processes is the proliferation of rat hepatocytes, so it can be used as a measure of the effectiveness of the method. Here we proposed an adaptive sparse group lasso to select genes in groups for rat hepatocyte proliferation. The weighted gene co-expression networks analysis was used to identify modules corresponding to gene pathways, based on which a strategy of dividing genes into groups was proposed. A strategy of adaptive gene selection was also presented by assessing the gene significance and introducing the adaptive lasso penalty. Moreover, an improved blockwise descent algorithm was proposed. Experimental results demonstrated that the proposed method can improve the classification accuracy, and select less number of significant genes which act jointly in groups and have direct or indirect effects on rat hepatocyte proliferation. The effectiveness of the method was verified by the method of rat hepatocyte proliferation.

Tmub1 negatively regulates liver regeneration via inhibiting STAT3 phosphorylation

Tmub1 (transmembrane and ubiquitin-like domain-containing 1) plays negative roles in rat hepatocyte proliferation, but its underlying molecular mechanisms in liver regeneration regulation have yet to be revealed. Here, we show that in vivo transfection of Tmub1 overexpression vectors impaired mouse liver regeneration after partial hepatectomy (PHx). Loss- and gain-of-function analyses in human hepatocyte Lo2 cells indicated that Tmub1 inhibits the phosphorylation of STAT3 and the activation of STAT3 signaling. Furthermore, the inhibitory effect of Tmub1 overexpression on hepatocyte proliferation can be reversed by the STAT3 activator OSM, while the promotive effect of Tmub1 knockdown can be abolished by the STAT3 inhibitor stattic. Coimmunoprecipitation assays revealed interaction between Tmub1 and STAT3. Finally, we present data from chromatin immunoprecipitation and luciferase reporter gene assays and report that STAT3 binds to and activates the promoter of Tmub1, suggesting a putative negative feedback loop between Tmub1 and STAT3 signaling. Taken together, the results of our study suggest that Tmub1 is an important negative regulator of hepatocyte proliferation in liver regeneration through STAT3 signaling. These findings provide a potential strategy for the management of liver regeneration.

Chlorogenic acid against palmitic acid in endoplasmic reticulum stress-mediated apoptosis resulting in protective effect of primary rat hepatocytes

BackgroundWe demonstrated growing evidence supports a protective role of chlorogenic acid of rat hepatocytes elicited by two compounds, i.e. thapsigargin and palmitic acid. Nevertheless, little is known about the mechanisms of palmitic acid induced endoplasmic reticulum (ER) stress and cell death.MethodsThe proliferation of primary rat hepatocytes was detected by MTT assay. The expression of GRP78, CHOP and GRP94 was detected by Western blot analyses.Caspase-3 activity was detected by a Caspase-3 substrate kit. Cell apoptosis was detected by Hoechst 33342 staining.ResultsWe demonstrated that incubation of hepatocytes for 16 h with palmitic acid elevated cell death. Moreover, Western blot analyses demonstrated increased levels of the endoplasmic reticulum stress markers — glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and glucose regulated protein 94 (GRP94). Chlorogenic acid could inhibit ER stress induced cell death and levels of indicators of ER stress caused by palmitic acid. The effect of thapsigargin, which evokes ER stress were reversed by chlorogenic acid.ConclusionsAltogether, our data indicate that in primary rat hepatocytes, chlorogenic acid prevents ER stress-mediated apoptosis of palmitic acid.

MiR-429 regulates rat liver regeneration and hepatocyte proliferation by targeting JUN/MYC/BCL2/CCND1 signaling pathway

Increasing evidence indicates that miR-429 is involved in tumor suppression in various human cancers. However, its role in liver regeneration remains unexplored. Liver regeneration is a highly orchestrated process that can be regulated by microRNAs (miRNAs), although the mechanisms are largely unclear. In this study, we aimed to identify the role of miR-429 in hepatocyte proliferation during liver regeneration. First, we performed microarray analysis and qRT-PCR. Results indicated that miR-429 level in rat liver markedly decreased 30 h after partial hepatectomy, and miR-429 overexpression disrupted BRL-3A proliferation and the transition of G1 to S phase in rat hepatocyte and promoted hepatocyte apoptosis. By contrast, miR-429 down-regulation had inverse effects. MiR-429 negatively regulated JUN expression in vitro and in vivo. After using JUN siRNA, we found that JUN inhibition mediates the effect of miR-429 in hepatocyte proliferation and growth and miR-429 negatively regulates JUN/MYC/BCL2/CCND1 signaling pathways. Our results also indicated that miR-429 inhibits hepatocyte proliferation and liver regeneration by targeting JUN/MYC/BCL2/CCND1.

PLCγ2 promotes apoptosis while inhibits proliferation in rat hepatocytes through PKCD/JNK MAPK and PKCD/p38 MAPK signalling.

The PLCG2 (PLCγ2) gene is a member of PLC gene family encoding transmembrane signalling enzymes involved in various biological processes including cell proliferation and apoptosis. Our earlier study indicated that PLCγ2 may be involved in the termination of regeneration of the liver which is mainly composed of hepatocytes, but its exact biological function and molecular mechanism in liver regeneration termination remains unclear. This study aims to examine the role of PLCγ2 in the growth of hepatocytes. A recombinant adenovirus expressing PLCγ2 was used to infect primary rat hepatocytes. PLCγ2 mRNA and protein levels were detected by qRT-PCR and Western blot. The subcellular location of PLCγ2 protein was tested by an immunofluorescence assay. The proliferation of hepatocytes was measured by MTT assay. The cell cycle and apoptosis were analysed by flow cytometry. Caspase-3, -8 and -9 activities were measured by a spectrophotometry method. Phosphorylation levels of PKCD, JNK and p38 in the infected cells were detected by Western blot. The possible mechanism underlying the role of PLCγ2 in hepatocyte growth was also explored by adding a signalling pathway inhibitor. Hepatocyte proliferation was dramatically reduced, while cell apoptosis was remarkably increased. The results demonstrated that PLCγ2 increased the phosphorylation of PKCD, p38 and JNK in rat hepatocytes. After PKCD activity was inhibited by the inhibitor Go 6983, the levels of both p-p38 and p-JNK MAPKs significantly decreased, and PLCγ2-induced cell proliferation inhibition and cell apoptosis were obviously reversed. This study showed that PLCγ2 regulates hepatocyte growth through PKCD-dependently activating p38 MAPK and JNK MAPK pathways; this result was experimentally based on the further exploration of the effect of PLCγ2 on hepatocyte growth in vivo.

Silencing of phospholipase C gamma 2 promotes proliferation of rat hepatocytes in vitro.

The management of hepatic failure is undoubtedly difficult, and poor results have led to the search for novel therapeutic approaches. Nowadays, anti-apoptotic gene therapy is considered as an ideal approach. It has been proved that phospholipase Cγ2 (PLCγ2) is involved in the apoptosis of immune cells and tumor cells; however, whether this gene is related to hepatocyte death is still unclear. This study examined the role of PLCγ2 by inhibiting its expression in rat hepatocytes with siRNA. We also further analyzed the cellular mechanism by which the expression inhibition of PLCγ2 induces cell death. Silencing PLCγ2 gene by adenovirus vector expressing PLCγ2-targeted siRNA caused the great decline in the number of G1- and G2/M phase cells, the significant increase in the number of S phase cells, and the obvious reduction in apoptosis index. In addition, silencing PLCγ2 gene relieved the rat hepatocyte damage, such as the cell shrinkage and chromatin condensation, nuclear fragmentation. Further analysis of Ad-PLCγ2 siRNA-transfected hepatocytes demonstrated that suppression of PLCγ2 gene expression could cause the caspase dependent cell death by inhibiting the signal pathway MEKK1/MKK4/JNK1/2/c-Jun. In conclusion, these findings suggest that interference with PLCγ2 expression could relieve the inhibitory effect of PLCγ2 on hepaocyte apoptosis, thus, promote proliferation through inactivating PKCδ-mediated JNK1/2 signaling pathway.

Effects of pharmacological inhibition of plasminogen binding on liver regeneration in rats.

The fibrinolysis system is thought to play an important role in liver regeneration. We previously found that plasminogen (Plg) is localized to the cell surface of regenerating liver tissue as well as proliferating hepatocytes in vitro. Here, we investigated the significance of Plg binding to the cell surface during liver regeneration. Pre-administration of tranexamic acid (TXA), which is a competitive inhibitor of Plg binding, to hepatectomized rats mildly delayed restoration of liver weight in vivo. Although binding of Plg to the cell membrane decreased following TXA administration, TXA showed little effect on hepatocyte proliferation in rats. We also discovered that Plg treatment did not stimulate proliferation of primary rat hepatocytes in vitro. These results suggest that Plg/plasmin potentiates liver regeneration via a pathway distinct from those through which hepatocyte proliferation is stimulated.

Upregulation of miR-223 in the rat liver inhibits proliferation of hepatocytes under simulated microgravity

Long-term spaceflight affects numerous organ systems in the body, including metabolic dysfunction. Recently, ample evidence has demonstrated that the liver is a vulnerable organ during spaceflight. However, the changes in hepatocyte proliferation and cell cycle control under microgravity remain largely unexplored. In the present study, we first confirmed that the serum levels of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, biochemical markers of liver function, were altered in rats under tail suspension (TS) conditions to simulate microgravity, as shown in previous reports. Next, we demonstrated that the cell proliferation activity, determined by Ki67, PCNA and PH3, was significantly decreased at the different TS time points (TS for 14, 28 and 42 days) compared with that in the control group. Consistently, the positive cell cycle regulators Ccna2, Ccnd1, Cdk1, Cdk2 and cyclin D3 were also significantly decreased in the TS groups as shown by quantitative real-time PCR and western blotting analysis. Subsequent analysis revealed that the aberrant hepatocyte proliferation inhibition under simulated microgravity was associated with the upregulation of miR-223 in the liver. We further found that miR-223 inhibited the proliferation of Hepa1-6 cells and identified CDK2 and CUL1 as its direct targets. In addition, the decreased expression of CDK2 and CUL1 was negatively correlated with the level of p27 in vitro and in vivo, which may have been responsible for retarding hepatocyte proliferation. Collectively, these data indicate that upregulation of miR-223 was associated with the inhibition of liver cell growth and reveal the role of miR-223 in rat hepatocyte proliferation disorders and the pathophysiological process under simulated microgravity.

Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation

Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2′-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.

MiRNA profiles in livers with different mass deficits after partial hepatectomy and miR-106b~25 cluster accelerating hepatocyte proliferation in rats.

Partial hepatectomy (PH) promotes the reentry of quiescent hepatocytes into cell cycle for regrowth. miRNA profiles in livers with different mass deficits after PH have not been investigated and miRNAs implicated in liver regeneration remain unclear. We generated miRNA profiles from normal and remnant livers at 6, 12, 24, and 36 hours after 1/3 or 2/3PH using microarrays. Compared with normal livers, the proportion of altered miRNAs decreased with time after 1/3PH, but increased after 2/3PH. Most of altered miRNAs between 1/3 and 2/3PH exhibited similar up- or down-regulation, but lower expression magnitude for 1/3PH. Among differentially expressed miRNAs between 2/3PH with robust DNA replication and 1/3PH with a minimal replicative response, we identified miR-101a, miR-92a, miR-25, miR-93 and miR-106b as key regulators of cell cycle. In 2/3PH model, overexpression of miR-106b~25 cluster tended to accelerate liver regeneration, while inhibition of miR-106b~25 cluster markedly repressed regenerative response and delayed recovery of liver function. Mechanistically, RB1 and KAT2B with cell cycle arrest activity were identified as novel targets of miR-106b/93 and miR-25, respectively. Overall, we featured miRNA profiles and dynamics after 1/3 and 2/3PH, and identified miR-106b~25 cluster as being involved in timely cell cycle entry of hepatocytes after PH.

Immunohistochemical and morphometric study of hepatocyte proliferation in 18-days old infant rats after a mechanical trauma being administered «Suvar» biologically active substance

Aim. Morphometric and immunohistochemical study of hepatocyte proliferative activity in 18-days-old infant rates after a mechanical trauma of the liver.&#x0D; Methods. After an artificial mechanical injury of the liver with a steel needle in 18-days old infant rats, a biologically active substance «Suvar» in a dose of 50 mg/kg (n=36) was administered to animals. After animals were withdrawn from the experiment, a morphometric study (where the number of mitoses of hepatocytes for every 7000 cells were calculated) and immunohistochemistry of liver tissue were performed. For the immunohistochemistry, two commercially available monoclonal antibodies kits (manufactured by «Santa Cruz») were used: (1) proliferative activity marker Ki-67; (2) apoptosis marker bcl-2. Immunohistochemistry was performed according to standard protocol. Thirty-six operated infant rats, in whom the biologically active substance was not administered, were analyzed as the controls.&#x0D; Results. The number of ki-67 positive hepatocytes in experimental animals on the 3rd, 5th,7th and the 9th day after the operation was significantly higher compared to controls. This was in accordance with the changes of the number of mitoses in hepatocytes, where the greatest number of mitoses was also registered on 3rd, 5th,7th and the 9th day. Both in experimental and in control infant rats, only single cells marked bcl-2 antibodies were found. The gained results support the opinion of the majority of the researchers that the liver regenerates mainly due to hepatocyte proliferation.&#x0D; Conclusion. Morphometric study and immunohistochemistry confirmed that biologically active substance «Suvar» stimulates hepatocyte proliferation of hepatic cells, allowing better healing of liver injury compared to control animals.

Disappearance of GFP-positive hepatocytes transplanted into the liver of syngeneic wild-type rats pretreated with retrorsine.

Background and AimGreen fluorescent protein (GFP) is a widely used molecular tag to trace transplanted cells in rodent liver injury models. The differing results from various previously reported studies using GFP could be attributed to the immunogenicity of GFP.MethodsHepatocytes were obtained from GFP-expressing transgenic (Tg) Lewis rats and were transplanted into the livers of wild-type Lewis rats after they had undergone a partial hepatectomy. The proliferation of endogenous hepatocytes in recipient rats was inhibited by pretreatment with retrorsine to enhance the proliferation of the transplanted hepatocytes. Transplantation of wild-type hepatocytes into GFP-Tg rat liver was also performed for comparison.ResultsAll biopsy specimens taken seven days after transplantation showed engraftment of transplanted hepatocytes, with the numbers of transplanted hepatocytes increasing until day 14. GFP-positive hepatocytes in wild-type rat livers were decreased by day 28 and could not be detected on day 42, whereas the number of wild-type hepatocytes steadily increased in GFP-Tg rat liver. Histological examination showed degenerative change of GFP-positive hepatocytes and the accumulation of infiltrating cells on day 28. PCR analysis for the GFP transgene suggested that transplanted hepatocytes were eliminated rather than being retained along with the loss of GFP expression. Both modification of the immunological response using tacrolimus and bone marrow transplantation prolonged the survival of GFP-positive hepatocytes. In contrast, host immunization with GFP-positive hepatocytes led to complete loss of GFP-positive hepatocytes by day 14.ConclusionGFP-positive hepatocytes isolated from GFP-Tg Lewis rats did not survive long term in the livers of retrorsine-pretreated wild-type Lewis rats. The mechanism underlying this phenomenon most likely involves an immunological reaction against GFP. The influence of GFP immunogenicity on cell transplantation models should be considered in planning in vivo experiments using GFP and in interpreting their results.

T3 receptor‐beta agonists GC‐1 (Sobetirome) and KB2115 (Eprotirome) stimulate hepatocyte proliferation in mice (649.5)

T3 hormone is known to be a strong inducer of hepatocyte proliferation in rats and mice. Recent work has shown that the molecular mechanism for this may be Protein Kinase A‐dependent activation of the β‐Catenin signaling pathway. This finding has shed light on the potential therapeutic use of thyroid hormone in select cases of liver failure. However, systemic side effects of T3 may preclude its clinical use. GC‐1 and KB2115 are T3 hormone analogs that bind selectively to the T3 hormone β‐receptor and do not seem to produce the undesirable cardiovascular effects. We investigate whether GC‐1 and KB2115 are able to induce proliferation in mouse hepatocytes. Nine C57/BL6 2‐month old male mice received eight daily intraperitoneal injections with GC‐1, KB2115, or DMSO (control) at 0.3mg/Kg body weight. Livers from mice injected with GC‐1 and KB2115 showed increased levels of Cyclin D‐1 on Western Blot compared with controls. We also saw an increase in Beta‐Catenin, Cyclin D‐1, and Ki‐67 by immunohistochemistry, without significant difference in serum biochemistry. This suggests that selective thyromimetics are likely activating β‐Catenin signaling pathway in a similar manner to T3 hormone, and with further study may prove useful as an adjunct therapeutic option in select cases of liver failure.Grant Funding Source: 1RO1DK62277 to SPM