Late Phase Of Liver Regeneration Research Articles

The integrated analysis of gut microbiota and metabolome revealed steroid hormone biosynthesis is a critical pathway in liver regeneration after 2/3 partial hepatectomy.

Introduction: The liver is the only organ capable of full regeneration in mammals. However, the exact mechanism of gut microbiota and metabolites derived from them relating to liver regeneration has not been fully elucidated. Methods: To demonstrate how the gut-liver axis contributes to liver regeneration, using an LC-QTOF/MS-based metabolomics technique, we examine the gut microbiota-derived metabolites in the gut content of C57BL/6J mice at various points after 2/3 partial hepatectomy (PHx). Compound identification, multivariate/univariate data analysis and pathway analysis were performed subsequently. The diversity of the bacterial communities in the gastrointestinal content was measured using 16S rRNA gene sequencing. Then, the integration analysis of gut microbiota and metabolome was performed. Results: After 2/3 PHx, the residual liver proliferated quickly in the first 3days and had about 90% of its initial weight by the seventh day. The results of PLS-DA showed that a significant metabolic shift occurred at 6h and 36h after 2/3 PHx that was reversed at the late phase of liver regeneration. The α and β-diversity of the gut microbiota significantly changed at the early stage of liver regeneration. Specifically, Escherichia Shigella, Lactobacillus, Akkermansia, and Muribaculaceae were the bacteria that changed the most considerably during liver regeneration. Further pathway analysis found the most influenced co-metabolized pathways between the host and gut bacteria including glycolysis, the TCA cycle, arginine metabolism, glutathione metabolism, tryptophan metabolism, and purine and pyrimidine metabolism. Specifically, steroid hormone biosynthesis is the most significant pathway of the host during liver regeneration. Discussion: These findings revealed that during liver regeneration, there was a broad modification of gut microbiota and systemic metabolism and they were strongly correlated. Targeting specific gut bacterial strains, especially increasing the abundance of Akkermansia and decreasing the abundance of Enterobacteriaceae, may be a promising beneficial strategy to modulate systemic metabolism such as amino acid and nucleotide metabolism and promote liver regeneration.

MicroRNA-125b-5p Regulates Hepatocyte Proliferation During the Termination Phase of Liver Regeneration.

The ability of the liver to regenerate and restore mass limits the increasing mortality rate due to life‐threatening liver diseases. Successful liver regeneration is accomplished in multiple stages, of which the priming and proliferation phases are well studied. However, the regulatory pathways, specifically microRNA (miRNA)‐mediated posttranscriptional regulation, which prevent uncontrolled proliferation and mediate the termination of liver regeneration, are not well understood. We identified differentially regulated miRNAs during the termination phase after 2/3 partial hepatectomy (PH) in mice, which is a well‐established mouse model of liver regeneration. We further evaluated the function of differentially regulated miRNAs in primary mouse hepatocytes by using mimics and inhibitors and in vivo by using adeno‐associated virus (AAV) serotype 8. A candidate miRNA target was identified by messenger RNA array in silico analyses and validated in primary mouse and human hepatocytes. Using miRNA profiling, we discovered miR‐125b‐5p as a novel regulator of hepatocyte proliferation in the late phase of liver regeneration. AAV‐mediated miR‐125b‐5p delivery in mice enhanced the endogenous regenerative capacity and resulted in improved restoration of liver mass after 2/3 PH. Further, we found that ankyrin repeat and BTB/POZ domain containing protein 1 (Abtb1) is a direct target of miR‐125b‐5p in primary mouse and human hepatocytes and contributes to the pro‐proliferative activity of miR‐125b‐5p by forkhead box G1 (FOXG1) and the cyclin‐dependent kinase inhibitor 1A (p21) pathway. Conclusion: miR‐125b‐5p has an important role in regulating hepatocyte proliferation in the termination phase of liver regeneration and may serve as a potential therapeutic target in various liver diseases that often exhibit deregulated hepatocyte proliferation.

Novel spheroid reservoir bioartificial liver improves survival of nonhuman primates in a toxin-induced model of acute liver failure.

This study aims to evaluate the effectiveness and safety of the spheroid reservoir bioartificial liver (SRBAL) with porcine hepatocyte organoids in a preclinical nonhuman primate model of acute liver failure (ALF).Methods: Thirty healthy rhesus monkeys were infused with α-amanitin and lipopolysaccharide and randomized into five groups (ALF alone control group; sham no-cell SRBAL treatment group; groups A, B and C with SRBAL treatment started at 12 h, 24 h and 36 h after induction of ALF, respectively). Animals were continuously treated with the SRBAL device for 6 h and followed for up to 336 h.Results: Survival of ALF monkeys improved with hepatocyte SRBAL treatment compared to control groups. Blood ammonia and total bilirubin were lower, and albumin levels were higher in all hepatocyte SRBAL treatment groups. No evidence of porcine endogenous retrovirus was identified in monkey liver or blood after SRBAL treatment. Titers of monkey antibody (IgG, IgM) did not rise after SRBAL treatment. In survival cases, the proportion of necrotic and apoptotic hepatocytes was lower in SRBAL-treated groups, with earlier liver regeneration leading to recovery. Cytokines TNF-α, IL-6, IL-12, IL-1β, IL-8, IFN-γ and IL-2 were ameliorated by the SRBAL treatment, while levels of M-CSF; HGF, EGF and VEGF; IL-1RA and MIF rose on priming, proliferation and the late phase of liver regeneration.Conclusions: The benefit of SRBAL therapy included preventive effects and therapeutic effects. SRBAL improved survival rate and prolonged median survival time in a nonhuman primate model of drug-induced ALF, and these benefits declined with a delay in the initiation of therapy. Improved survival and recovery of ALF monkeys was associated with a reduction in blood ammonia levels, inhibition of the pro-inflammatory response of ALF, and provided a microenvironment more suitable for regeneration of the injured liver.

Transcriptome Temporal and Functional Analysis of Liver Regeneration Termination

Decades of liver regeneration studies still left the termination phase least elucidated. However regeneration ending mechanisms are clinicaly relevant. We aimed to analyse the timing and transcriptional control of the latest phase of liver regeneration, both controversial. Male Wistar rats were subjected to 2/3 partial hepatectomy with recovery lasting from 1 to 14 days. Time-series microarray data were assessed by innovative combination of hierarchical clustering and principal component analysis and validated by real-time RT-PCR. Hierarchical clustering and principal component analysis in agreement distinguished three temporal phases of liver regeneration. We found 359 genes specifically altered during late phase regeneration. Gene enrichment analysis and manual review of microarray data suggested five pathways worth further study: PPAR signalling pathway; lipid metabolism; complement, coagulation and fibrinolytic cascades; ECM remodelling and xenobiotic biotransformation. Microarray findings pertinent for termination phase were substantiated by real-time RT-PCR. In conclusion, transcriptional profiling mapped late phase of liver regeneration beyond 5(th) day of recovery and revealed 5 pathways specifically acting at this time. Inclusion of longer post-surgery intervals brought improved coverage of regeneration time dynamics and is advisable for further works. Investigation into the workings of suggested pathways might prove helpful in preventing and managing liver tumours.

Iron-regulatory gene expression during liver regeneration

Background. In rat, the first 18–24 h after partial hepatectomy (PH) are characterized by an acute-phase reaction, after which liver regeneration predominates. Interleukin-6 (IL-6) induces the iron hormone hepcidin, which blocks iron uptake and may compromise iron uptake in the growing liver. The expressions of hepcidin and the iron-regulatory pathway of hepcidin gene expression during the late phase of liver regeneration are unknown. Aim. To characterize the expression pattern of hepcidin and the iron-sensing pathway of hepcidin regulation during liver regeneration. Methods. Rats were subjected to PH or sham operation. Liver weights, number of S-phase nuclei, and serum levels of iron and IL-6 were determined. Messenger-RNA levels of hepcidin, ferritin, hemojuvelin, transferrin receptor 1 and 2, HFE, divalent metal transporter 1, ferroportin, and ceruloplasmin were determined with qPCR at different time points. Protein levels of STAT3 and SMAD4 were determined with western blot. Results. During the acute-phase response, IL-6 release induced STAT3 protein and hepcidin mRNA, whereas mRNA levels of proteins in the iron-sensing pathway (HFE, hemojuvelin, and transferrin receptor 2) decreased. The mRNA levels of proteins involved in cellular iron uptake were increased and cellular iron export unchanged. During liver regeneration >24 h after PH, gene expressions in the iron-sensing pathway were continuously suppressed and hepcidin mRNA levels declined 3–7 days after surgery. Conclusions. Hepcidin gene expression peaks during the acute-phase response, but a sustained down-regulation of the iron-sensing pathway of hepcidin regulation gradually reduces hepcidin gene expression until regeneration is complete, thereby promoting iron mobilization to the regenerating liver.

Eight paths of ERK1/2 signalling pathway regulating hepatocyte proliferation in rat liver regeneration

Although it is known that hormones, growth factors and integrin promote hepatocyte proliferation in liver regeneration (LR) through ERK1/2 signalling pathway, reports about regulating processes of its intracellular paths in hepatocytes of LR are limited. This study aims at exploring which paths of ERK1/2 signalling pathway participate in the regulation of rat LR, especially in hepatocyte proliferation, and how they do so. In all, 14 paths and 165 genes are known to be involved in ERK1/2 signalling pathway. Of them, 161 genes are included in Rat Genome 230 2.0 Array. This array was used to detect expression changes of genes related to ERK1/2 signalling pathway in isolated hepatocytes of rat LR, showing that 60 genes were related to hepatocytes of LR. In addition, bioinformatics and systems biology methods were used to analyse the roles of 14 above paths in regenerating hepatocytes. We found that three paths, RTK→SHC→GRB2/SOS→RAS→RAF, IntegrinΒ→FAK→RAC→PAK→RAF and GΒγ→PI3KΒγ→RAC→PAK→RAF, promoted the G1 phase progression of hepatocytes by activating ERK1/2. A further four paths, Gq→PLCΒ→PKC→SRC/PYK2→GRB2/SOS→RAS→RAF, RTK→PLCγ→PKC→SRC/PYK2→GRB2/SOS→RAS→RAF, IntegrinΒ→FAK/SRC→GRB2/SOS→RAS→RAF and IntegrinΒ→FAK→RAC→PAK→RAF, advanced the cell progression of S phase and G(2)/M checkpoint by activating ERK1/2, and so did PP1/2→Mek1/2 by decreasing the negative influence on ERK1/2. At the late phase of LR, Gαs→AC→EPAC→Rap1→Raf blocked hepatocyte proliferation by decreasing the activity of ERK1/2 and so did PP1/2→Mek1/2. In summary, 60 genes and 8 paths of ERK1/2 signalling pathway regulated hepatocyte proliferation in rat LR.

Mir-34a Is Upregulated during Liver Regeneration in Rats and Is Associated with the Suppression of Hepatocyte Proliferation

BackgroundMicroRNAs are a class of small regulatory RNAs that modulate a variety of biological processes, including cellular differentiation, apoptosis, metabolism and proliferation. This study aims to explore the effect of miR-34a in hepatocyte proliferation and its potential role in liver regeneration termination.Methodology/Principal FindingMiR-34a was highly induced after partial hepatectomy. Overexpression of miR-34a in BRL-3A cells could significantly inhibit cell proliferation and down-regulate the expression of inhibin βB (INHBB) and Met. In BRL-3A cells, INHBB was identified as a direct target of miR-34a by luciferase reporter assay. More importantly, INHBB siRNA significantly repressed cell proliferation. A decrease of INHBB and Met was detected in regenerating liver.Conclusion/SignificanceMiR-34a expression was upregulated during the late phase of liver regeneration. MiR-34a-mediated regulation of INHBB and Met may collectively contribute to the suppression of hepatocyte proliferation.

Lipid Metabolism and Peroxisome Proliferator-Activated Receptor Signaling Pathways Participate in Late-Phase Liver Regeneration

Liver regeneration (LR) is of great clinical significance in various liver-associated diseases. LR proceeds along a sequence of three distinct phases: priming/initiation, proliferation, and termination. Compared with the recognition of the first two phases, little is known about LR termination and structure/function reorganization. A combination of "omics" techniques, along with bioinformatics, may provide new insights into the molecular mechanism of the late-phase LR. Gene, protein, and metabolite profiles of the rat liver were determined by cDNA microarray, two-dimensional electrophoresis, and HPLC-MS analysis. Pathway enrichment analysis was performed to identify the pathways: 427 differentially expressed genes extracted from the microarray experiment revealed two expression patterns representing the early and late phase of LR. Functionally, the genes expressing at a higher level at the early phase than at the late phase were mainly involved in the response to stress, proliferation, and resistance to apoptosis, while those expressing at a lower level at the early phase than at the late phase were mainly engaged in lipid metabolism. Compared with the sham-operation control (SH) group, 5 proteins in the 70% partial hepatectomy (70%PHx) group were upregulated at the protein level, and 3 proteins were downregulated at 168 h after the 70%PHx. E-FABP, an upregulated fatty acid binding protein, was found to be involved in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. The metabolomic data confirmed the enhancement of lipid metabolism by the detection of the intermediate and final metabolites. We've concluded that increased lipid metabolism and activated PPAR signaling pathways play important roles in late-phase LR.

Role of peroxisome proliferator‐activated receptor‐gamma (PPARγ) during liver regeneration in rats

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear receptor superfamily, is widely expressed in adipocytes and other tissues, including the liver. Several reports have shown that PPARgamma activation induced cell-cycle arrest and apoptosis in tumor cells. We investigated the role of the PPARgamma/ligand system and the effect of the PPARgamma agonist during liver regeneration. Expression of PPARgamma and serum levels of 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by enzyme immunoassay were evaluated in rats following partial hepatectomy (PH group). Further, the effect of the PPARgamma agonist, pioglitazone, on liver regeneration (PH + PGZ group) was evaluated by proliferating cell nuclear antigen labeling index, relative liver weight, and expression of cell-cycle regulators. The number of PPARgamma-stained hepatocytes decreased at 24 h (PH, 15.8 +/- 2.2%; sham, 35.5 +/- 2.4%; P < 0.001) and increased in the late phase of liver regeneration compared to the sham-operated group (P < 0.001 at 48-120 h). The peaks of serum 15d-PGJ2 (627.0 +/- 91.1 pg/ml) and PPARgamma expression (90.6 +/- 3.1%) coincided in the late phase of liver regeneration. Also, oral administration of pioglitazone inhibited hepatocyte proliferation, in terms of the proliferating cell nuclear antigen (PCNA) labeling index and p27 expression during the late phase of liver regeneration, and caused a transient reduction in liver mass when compared to the PH group. These results indicate that the PPARgamma/ligand system may be one of the key negative regulators of hepatocyte proliferation and may be responsible for the inhibition of liver growth in the late phase of liver regeneration.

Liver Regeneration Stimulates Tumor Metastases

Partial hepatectomy for patients with colorectal liver metastases is associated with a tumor recurrence rate approaching 80% post-resection. Different factors and phases associated with regeneration of the liver are implicated in tumor recurrence. This study investigates the effects of the early and late phases of liver regeneration and the impact of the degree of liver resection on stimulating tumor growth and metastasis. Groups of mice underwent partial hepatectomy (37% or 70%) and were then challenged with colorectal liver carcinoma (CRC) tumors immediately after liver resection (early and late phase effect) or 6 days post liver resection (late phase effect). Tumor growth, degree of proliferation, tumor morphology, and the presence of extrahepatic metastases were investigated 21 days post-tumor induction. The late phase of liver regeneration plays a significant role in tumor stimulation and metastasis. The degree of hepatectomy also appears to be an important factor. The degree of hepatic resection significantly influences tumor growth and the extent of extrahepatic metastases, particularly in the lungs. Elucidation of the processes involved in the late phase of liver regeneration may assist in the development and timing of adjuvant agents to minimize tumor recurrence during this phase.

Epimorphin expression and stellate cell status in mouse liver injury

Epimorphin, a mesenchymal morphogenic protein expressed by hepatic stellate cells, is considered important to liver morphogenesis in both healthy and pathologic conditions. However, the stellate cell phenotype, quiescent versus activated, that expresses epimorphin is unknown. We studied the relationship between epimorphin expression and stellate cell status in carbon tetrachloride-induced acute and chronic injury to mouse liver and in mouse liver regeneration following 70% partial hepatectomy. Epimorphin-positive cells in sinusoids expressed desmin, indicating that they are stellate cells. Epimorphin-positive cells were more numerous and larger in pericentral than periportal sinusoids in normal liver. In early-phase acute liver injury and liver regeneration, epimorphin expression transiently decreased while alphaSMA-positive stellate cells increased. In the recovery phase of acute and chronic injury as well as the late phase of liver regeneration, epimorphin expression was strikingly enhanced while alphaSMA-positive stellate cells decreased. This expression pattern was seen in both Balb/c and C57BL6 mouse strains irrespective of their differences in response to the hepatotoxin. In conclusion, stellate cells express epimorphin in their quiescent state and in the recovery phase, respectively associated with maintenance and reconstruction of microscopic liver structure.

Late phase of liver restoration following partial hepatectomy in phenobarbital-treated rats. II. Effect of phenobarbital on aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, and UDP-glucuronyltransferase activity.

Rats were treated with 50 mg phenobarbital (PB) per kg body weight for 7 days prior to or after partial hepatectomy. The activities of aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase were measured in the regenerating liver 1 week following liver amputation. UDP-glucuronyl transferase activity was determined at the time of surgery as well as 7 days later at the time of death. Alanine aminotransferase was induced by PB in rats only treated in the postoperative period, while aspartate aminotransferase and glutamate dehydrogenase were not. The activity of UDP-glucuronyl transferase was increased more than twofold by repeated PB treatment in both normal and regenerating liver. After cessation of therapy, however, the enhanced activities returned to a normal level. It is concluded that UDP-glucuronyl transferase activity in regenerating liver is as inducible as in normal rat liver by repeated PB treatment despite incomplete hepatic regeneration. Preoperative PB treatment alone is not sufficient to stimulate the glucuronylating pathway in the late phase of liver regeneration.