Stimulates Hepatocyte Proliferation Research Articles

TRIM26 deficiency enhancing liver regeneration through macrophage polarization and β-catenin pathway activation

Liver regeneration is a complex process involving the crosstalk between parenchymal and non-parenchymal cells, especially macrophages. However, the underlying mechanisms remain incompletely understood. Here, we identify the E3 ubiquitin ligase TRIM26 as a crucial regulator of liver regeneration. Following partial hepatectomy or acute liver injury induced by carbon tetrachloride, Trim26 knockout mice exhibit enhanced hepatocyte proliferation compared to wild-type controls, while adeno-associated virus (AAV)-mediated overexpression of Trim26 reverses the promotional effects. Mechanistically, Trim26 deficiency promotes the recruitment of macrophages to the liver and their polarization towards pro-inflammatory M1 phenotype. These M1 macrophages secrete Wnts, including Wnt2, which subsequently stimulate hepatocyte proliferation through the activation of Wnt/β-catenin signaling. In hepatocytes, Trim26 knockdown reduces the ubiquitination and degradation of β-catenin, thereby further enhancing Wnt/β-catenin signaling. Pharmacological inhibition of Wnt/β-catenin pathway by ICG-001 or depletion of macrophages by clodronate liposomes diminishes the pro-regenerative effects of Trim26 deficiency. Moreover, bone marrow transplantation experiments provide evidence that Trim26 knockout in myeloid cells alone can also promote liver regeneration, highlighting the critical role of macrophage Trim26 in this process. Taken together, our study uncovers TRIM26 as a negative regulator of liver regeneration by modulating macrophage polarization and Wnt/β-catenin signaling in hepatocytes, providing a potential therapeutic target for promoting liver regeneration in clinical settings.

Phosphatidic acid-enabled MKL1 contributes to liver regeneration: Translational implication in liver failure

Liver regeneration following injury aids the restoration of liver mass and the recovery of liver function. In the present study we investigated the contribution of megakaryocytic leukemia 1 (MKL1), a transcriptional modulator, to liver regeneration. We report that both MKL1 expression and its nuclear translocation correlated with hepatocyte proliferation in cell and animal models of liver regeneration and in liver failure patients. Mice with MKL1 deletion exhibited defective regenerative response in the liver. Transcriptomic analysis revealed that MKL1 interacted with E2F1 to program pro-regenerative transcription. MAPKAPK2 mediated phosphorylation primed MKL1 for its interaction with E2F1. Of interest, phospholipase d2 promoted MKL1 nuclear accumulation and liver regeneration by catalyzing production of phosphatidic acid (PA). PA administration stimulated hepatocyte proliferation and enhanced survival in a MKL1-dependent manner in a pre-clinical model of liver failure. Finally, PA levels was detected to be positively correlated with expression of pro-regenerative genes and inversely correlated with liver injury in liver failure patients. In conclusion, our data reveal a novel mechanism whereby MKL1 contributes to liver regeneration. Screening for small-molecule compounds boosting MKL1 activity may be considered as a reasonable approach to treat acute liver failure.

EpCAM and APN expression in combination with γ-H2AX as biomarkers for detecting hepatocarcinogens in rats.

The phosphorylated form of histone H2AX (γ-H2AX) serves as a commonly utilized biomarker for DNA damage. Based on our previous findings, which demonstrated the formation of γ-H2AX foci as a reliable biomarker for detecting bladder carcinogens in repeated dose 28-day study in rats, we hypothesized that γ-H2AX could also function as a biomarker for detecting hepatocarcinogens. However, we found that γ-H2AX foci formation was not effectively induced by hepatocarcinogens that did not stimulate hepatocyte proliferation. Therefore, we explored alternative biomarkers to detect chemical hepatocarcinogenicity and discovered increased expressions of epithelial cell adhesion molecule (EpCAM/CD326)- and aminopeptidase N (APN/CD13) in the hepatocytes of rats administered various hepatocarcinogens. Significant increases in EpCAM- and APN-positive hepatocytes were observed for eight and five of the 10 hepatocarcinogens, respectively. Notably, five and two of them, respectively, were negative for γ-H2AX foci. These results highlight the potential of EpCAM and APN as useful biomarkers in combination with γ-H2AX for the detection of chemical hepatocarcinogenicity.

Senescent hepatic stellate cells promote liver regeneration through IL-6 and ligands of CXCR2.

Senescent cells have long been associated with deleterious effects in aging-related pathologies, although recent studies have uncovered their beneficial roles in certain contexts, such as wound healing. We have found that hepatic stellate cells (HSCs) underwent senescence within 2 days after 2/3 partial hepatectomy (PHx) in young (2–3 months old) mice, and the elimination of these senescent cells by using the senolytic drug ABT263 or by using a genetic mouse model impaired liver regeneration. Senescent HSCs secrete IL-6 and CXCR2 ligands as part of the senescence-associated secretory phenotype, which induces multiple signaling pathways to stimulate liver regeneration. IL-6 activates STAT3, induces Yes-associated protein (YAP) activation through SRC family kinases, and synergizes with CXCL2 to activate ERK1/2 to stimulate hepatocyte proliferation. The administration of either IL-6 or CXCL2 partially restored liver regeneration in mice with senescent cell elimination, and the combination of both fully restored liver weight recovery. Furthermore, the matricellular protein central communication network factor 1 (CCN1, previously called CYR61) was rapidly elevated in response to PHx and induced HSC senescence. Knockin mice expressing a mutant CCN1 unable to bind integrin α6β1 were deficient in senescent cells and liver regeneration after PHx. Thus, HSC senescence, largely induced by CCN1, is a programmed response to PHx and plays a critical role in liver regeneration through signaling pathways activated by IL-6 and ligands of CXCR2.

Canonical Thyroid Hormone Receptor β Action Stimulates Hepatocyte Proliferation in Male Mice.

3,5,3'-L-triiodothyronine (T3) is a potent inducer of hepatocyte proliferation via the Wnt/β-catenin signaling pathway. Previous studies suggested the involvement of rapid noncanonical thyroid hormone receptor (TR) β signaling, directly activating hepatic Wnt/β-catenin signaling independent from TRβ DNA binding. However, the mechanism by which T3 increases Wnt/β-catenin signaling in hepatocytes has not yet been determined. We aimed to determine whether DNA binding of TRβ is required for stimulation of hepatocyte proliferation by T3. Wild-type (WT) mice, TRβ knockout mice (TRβ KO), and TRβ mutant mice with either specifically abrogated DNA binding (TRβ GS) or abrogated direct phosphatidylinositol 3 kinase activation (TRβ 147F) were treated with T3 for 6 hours or 7 days. Hepatocyte proliferation was assessed by Kiel-67 (Ki67) staining and apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Activation of β-catenin signaling was measured in primary murine hepatocytes. Gene expression was analyzed by microarray, gene set enrichment analysis (GSEA), and quantitative reverse transcription polymerase chain reaction. T3 induced hepatocyte proliferation with an increased number of Ki67-positive cells in WT and TRβ 147F mice (9.2% ± 6.5% and 10.1% ± 2.9%, respectively) compared to TRβ KO and TRβ GS mice (1.2% ± 1.1% and 1.5% ± 0.9%, respectively). Microarray analysis and GSEA showed that genes of the Wnt/β-catenin pathway-among them, Fzd8 (frizzled receptor 8) and Ctnnb1 (β-catenin)-were positively enriched only in T3-treated WT and TRβ 147F mice while B-cell translocation gene anti-proliferation factor 2 was repressed. Consequently, expression of Ccnd1 (CyclinD1) was induced. Instead of directly activating Wnt signaling, T3 and TRβ induce key genes of the Wnt/β-catenin pathway, ultimately stimulating hepatocyte proliferation via CyclinD1. Thus, canonical transcriptional TRβ action is necessary for T3-mediated stimulation of hepatocyte proliferation.

Chorionic gonadotropin stimulates maternal hepatocyte proliferation during pregnancy

The liver increases its size during pregnancy to adapt to metabolic demand associated with pregnancy. Our previous study showed that proliferation of maternal hepatocytes are increased during pregnancy in mice and that estradiol (E2) is one of the candidate hormones responsible for maternal hepatocyte proliferation. Here, we discovered that chorionic gonadotropin (CG) induces maternal hepatocyte proliferation during pregnancy. CG administration was sufficient to stimulate hepatocyte proliferation in non-pregnant mice as well as in cell culture system. We conclude that CG stimulates proliferation in the early pregnancy of maternal hepatocytes. In contrast, estrogen stimulates hepatocyte proliferation in the late pregnancy.

Bile Salt and FGF19 Signaling in the Early Phase of Human Liver Regeneration.

The involvement of bile salt–fibroblast growth factor 19 (FGF19) signaling in human liver regeneration (LR) is not well studied. Therefore, we studied aspects of bile salt–FGF19 signaling shortly after liver resection in patients. We compared plasma bile salt and FGF19 levels in arterial, portal and hepatic venous blood, calculated venous‐arterial differences (ΔVA), and determined hepatic transcript levels on two intra‐operative time points: before (< 1 hour) and immediately after (> 2‐3 hours) liver resection (i.e., following surgery). Postoperative bile salt and FGF19 levels were assessed on days 1, 2, and 3. LR was studied by computed tomography (CT)–liver volumetry. Following surgery, the liver, arterial, and portal bile salt levels were elevated (P < 0.05). Furthermore, an increased amount of bile salts was released in portal blood and extracted by the remnant liver (P < 0.05). Postoperatively, bile salt levels were elevated from day 1 onward (P < 0.001). For FGF19, intra‐operative or postoperative changes of ΔVA or plasma levels were not observed. The bile salt–homeostatic regulator farnesoid X receptor (FXR) was markedly up‐regulated following surgery (P < 0.001). Cell‐cycle re‐entry priming factors (interleukin 6 [IL‐6], signal transducer and activator of transcription 3 [STAT3], and cJUN) were up‐regulated following surgery and were positively correlated with FXR expression (P < 0.05). Postoperative hyperbilirubinemia was preceded by postsurgery low FXR and high Na+/Taurocholate cotransporting polypeptide (NTCP) expression in the remnant liver coupled with higher liver bile salt content (P < 0.05). Finally, bile salt levels on postoperative day 1 were an independent predictor of LR (P < 0.05). Conclusion: Systemic, portal, and liver bile salt levels are rapidly elevated after liver resection. Postoperative bile salts were positively associated with liver volume gain. In the studied time frame, FGF19 levels remained unaltered, suggesting that FGF19 plays a minor role in human LR. These findings indicate a more relevant role of bile salts in human LR.

Hepatitis B Virus DNA Integration and Clonal Expansion of Hepatocytes in the Chronically Infected Liver.

Human hepatitis B virus (HBV) can cause chronic, lifelong infection of the liver that may lead to persistent or episodic immune-mediated inflammation against virus-infected hepatocytes. This immune response results in elevated rates of killing of virus-infected hepatocytes, which may extend over many years or decades, lead to fibrosis and cirrhosis, and play a role in the high incidence of hepatocellular carcinoma (HCC) in HBV carriers. Immune-mediated inflammation appears to cause oxidative DNA damage to hepatocytes, which may also play a major role in hepatocarcinogenesis. An additional DNA damaging feature of chronic infections is random integration of HBV DNA into the chromosomal DNA of hepatocytes. While HBV DNA integration does not have a role in virus replication it may alter gene expression of the host cell. Indeed, most HCCs that arise in HBV carriers contain integrated HBV DNA and, in many, the integrant appears to have played a role in hepatocarcinogenesis. Clonal expansion of hepatocytes, which is a natural feature of liver biology, occurs because the hepatocyte population is self-renewing and therefore loses complexity due to random hepatocyte death and replacement by proliferation of surviving hepatocytes. This process may also represent a risk factor for the development of HCC. Interestingly, during chronic HBV infection, hepatocyte clones detected using integrated HBV DNA as lineage-specific markers, emerge that are larger than those expected to occur by random death and proliferation of hepatocytes. The emergence of these larger hepatocyte clones may reflect a survival advantage that could be explained by an ability to avoid the host immune response. While most of these larger hepatocyte clones are probably not preneoplastic, some may have already acquired preneoplastic changes. Thus, chronic inflammation in the HBV-infected liver may be responsible, at least in part, for both initiation of HCC via oxidative DNA damage and promotion of HCC via stimulation of hepatocyte proliferation through immune-mediated killing and compensatory division.

Bacterial hepatocyte growth factor receptor agonist stimulates hepatocyte proliferation and accelerates liver regeneration in a partial hepatectomy rat model.

Hepatocyte growth factor (HGF) is central to liver regeneration. The Internalin B (InlB) protein is a virulence factor produced by the pathogenic bacterium Listeria monocytogenes. InlB is known to mimic HGF activity by interacting with the HGF receptor (HGFR) and activating HGFR-controlled signaling pathways. We expressed and purified the HGFR-binding InlB domain, InlB321/15, cloned from the fully virulent clinical L. monocytogenes strain. HGFR and Erk1/2 phosphorylation was determined using Western blotting. The capacity of InlB321/15 to bind HGFR was measured using microscale thermophoresis. Liver regeneration was studied in a model of 70% partial hepatectomy (70%PHx) in male Wistar rats. The nuclear grade parameters were quantified using manual (percentage of binuclear hepatocytes), automated (nuclear diameters), or combined (Ki67 proliferation index) scoring methods. Purified InlB321/15 stimulated HGFR and Erk1/2 phosphorylation and accelerated the proliferation of HepG2 cells. InlB321/15 bound HGFR with Kd = 7.4 ± 1.3 nM. InlB321/15 injected intravenously on the second, fourth, and sixth days after surgery recovered the liver mass and improved the nuclear grade parameters. Seven days post 70% PHx, the liver weight indexes were 2.9 and 2.0%, the hepatocyte proliferation indexes were 19.8 and 0.6%, and the percentages of binucleated hepatocytes were 6.7 and 4.0%, in the InlB321/15-treated and control animals, respectively. Obtained data demonstrated that InlB321/15 improved hepatocyte proliferation and stimulated liver regeneration in animals with 70% hepatectomy.

Potential role of two novel agonists of thyroid hormone receptor-β on liver regeneration.

ObjectivesAlthough the hepatomitogenic activity of triiodothyronine (T3) is well established, the wide range of harmful effects exerted by this hormone precludes its use in liver regenerative therapy. Selective agonists of the beta isoform of thyroid hormone receptor (TRβ) do not exhibit T3‐induced cardiotoxicity and show a good safety profile in patients with NASH. The aim of this study was to investigate whether two novel TRβ agonists, the prodrug TG68 and the active compound IS25 could stimulate hepatocyte proliferation without T3/TRα‐dependent side effects.MethodsRats were treated with three different doses (12.5, 25 and 50 μg/100 g body weight) for one week. Hepatocyte proliferation, liver injury and serum biochemical parameters were measured by immunohistochemistry, qRT‐PCR and Western blot.ResultsBoth drugs increased hepatocyte proliferation as assessed by bromodeoxyuridine incorporation (from 14% to 28% vs 5% of controls) and mitotic activity. Enhanced proliferation occurred in the absence of significant signs of liver injury as shown by lack of increased serum transaminase levels or of apoptosis. No cardiac or renal hypertrophy typically associated with treatment with T3 was observed. Importantly, no proliferation of pancreatic acinar cells, such as that seen after administration of T3 or the TRβ agonist GC1 was detected following either TG68 or IS25, demonstrating the hepato‐specificity of these novel TRβ agonists.ConclusionsThe present study shows that TG68 and IS25 induce massive hepatocyte proliferation without overt toxicity. Hence, these agents may have a significant clinical application for regenerative therapies in liver transplantation or other surgical settings.

MiR-222 regulates proliferation of primary mouse hepatocytes in vitro

It is well known that hepatocytes regenerate after liver injury, although it is difficult to reproduce this phenomenon in vitro. The goal of this research was to determine the factors that stimulate proliferation of primary mouse hepatocytes (PMHs) in vitro. We first tested knockdown (KD) of tumor protein 53 (p53) alone as well as partial hepatectomy (PH, performed 72 h prior to PMHs preparation) alone. However, neither intervention stimulated hepatocyte proliferation during the 72-h observation period in vitro. We then tested the combination of p53 KD with PH and found that these interventions together stimulated cell proliferation in vitro. Under these latter conditions we analyzed gene expression of these cells by mRNA sequencing (RNA-seq) and microRNA sequencing (miRNA-seq). TargetScan analysis, which determines the relationship between microRNAs and gene expression, found a relationship between downregulated mmu-mir-222 (miR-222) and upregulated genes such as mitogen-activated protein kinase kinase kinase 2 (Map3k2). To confirm this relationship, we performed miR-222 KD and overexpression (OE) and observed the expected changes in target gene expression. Furthermore, the finding that miR-222 KD or OE stimulates or suppresses, respectively, hepatocyte proliferation is well explained by the association between miR-222 and its target genes, which stimulate growth. Our results suggest that miR-222 is one of the key factors regulating PMH proliferation in vitro.

Hepatic cell sheets engineered from human mesenchymal stem cells with a single small molecule compound IC-2 ameliorate acute liver injury in mice.

IntroductionWe previously reported that transplantation of hepatic cell sheets from human bone marrow-derived mesenchymal stem cells (BM-MSCs) with hexachlorophene, a Wnt/β-catenin signaling inhibitor, ameliorated acute liver injury. In a further previous report, we identified IC-2, a newly synthesized derivative of the Wnt/β-catenin signaling inhibitor ICG-001, as a potent inducer of hepatic differentiation of BM-MSCs.MethodsWe manufactured hepatic cell sheets by engineering from human BM-MSCs using the single small molecule IC-2. The therapeutic potential of IC-2-induced hepatic cell sheets was assessed by transplantation of IC-2- and hexachlorophene-treated hepatic cell sheets using a mouse model of acute liver injury.ResultsSignificant improvement of liver injury was elicited by the IC-2-treated hepatic cell sheets. The expression of complement C3 was enhanced by IC-2, followed by prominent hepatocyte proliferation stimulated through the activation of NF-κB and its downstream molecule STAT-3. Indeed, IC-2 also enhanced the expression of amphiregulin, resulting in the activation of the EGFR pathway and further stimulation of hepatocyte proliferation. As another important therapeutic mechanism, we revealed prominent reduction of oxidative stress mediated through upregulation of the thioredoxin (TRX) system by IC-2-treated hepatic cell sheets. The effects mediated by IC-2-treated sheets were superior compared with those mediated by hexachlorophene-treated sheets.ConclusionThe single compound IC-2 induced hepatic cell sheets that possess potent regeneration capacity and ameliorate acute liver injury.

Administration of Ginkgo biloba Extract (EGb761) Alone and in Combination with FK506 Promotes Liver Regeneration in a Rat Model of Partial Hepatectomy

Background:Free radical damage is known to occur during liver regeneration. The Ginkgo biloba extract EGb761 has antioxidant properties due to its ability to scavenge free radicals. FK506 has been widely used as an immunosuppressant that stimulates hepatocyte proliferation following partial hepatectomy.Aims:To explore whether EGb761 enhances liver regeneration after hepatectomy in rats, we investigated the effects of EGb761 alone and in combination with FK506 on the liver regenerative process.Study Design:Animal experimentation.Methods:A total of 75 Wistar albino rats weighing 340.08±11.66 g were randomly divided into five experimental groups: sham, control, FK506, EGb761, and FK506 + EGb761. According to the study groups, rats were administered FK506 at a dose of 0.1 mg/kg/day and EGb761 at 25 mg/kg/day three times via the intraperitoneal route. Then, two-thirds hepatectomy was performed according to the Higgins and Anderson technique in all the rats. At postoperative 48 h, 53 surviving rats were sacrificed. Serum and plasma samples were collected for analyzing thymidine kinase and oxidative stress marker levels. The regenerated liver was entirely resected, weighed, and sectioned. The mitotic index was assessed using hematoxylin-eosin staining. The extent of liver regeneration was calculated using the Child’s formula. The data were statistically analyzed using ANOVA, with a significance level of 5% (p<0.05).Results:Rats who received EGb761 showed significantly higher levels of liver regeneration than those who received FK506 or FK506 + EGb761 (p<0.01). Thymidine kinase level and mitotic index were significantly higher in the EGb761 (p<0.005) and FK506 (p<0.05) groups than in the control and sham groups. In addition, the liver regeneration percentage was significantly higher in the EGb761 group than in the FK506 group (p<0.01). Myeloperoxidase and malondialdehyde levels were significantly correlated between the EGb761 and FK506 groups, even at lower levels in the EGb761 group (p<0.001).Conclusion:EGb761, which is an antioxidant, reduces liver damage and stimulates liver regeneration following partial hepatectomy in rats through its anti-inflammatory and antioxidative effects.

Ghrelin Stimulates Hepatocyte Proliferation via Regulating Cell Cycle Through GSK3β/Β-Catenin Signaling Pathway

Background/Aims: Obesity is associated with a reduction in ghrelin, a 28 aa gastric hormone. Whether reduced ghrelin contributes to the impaired proliferation of hepatocytes associated with obesity-related steatosis remains largely unknown. Here we examined the effects of ghrelin on the proliferation of hepatocytes derived from lean and obese mice. Methods: AML 12 cells or hepatocytes isolated from mice fed normal chow diet (NCD) or high fat diet (HFD) were used. Effects of ghrelin on hepatocyte proliferation were detected with CCK8 assay and EdU staining. Cell cycle was analyzed by flow cytometry. Levels of proliferation markers was examined by Western blot. Results: Growth hormone secretagogue receptor 1a (GHS-R1a) mRNA and protein were present in hepatocytes. Levels of GHS-R1a were increased upon ghrelin treatment. Ghrelin significantly increased hepatocyte proliferation measured by Cell Counting Kit-8(CCK8) assay and EdU staining in a dose- and time-dependent manner. Proportion of cells in S phase was markedly increased upon treatment with ghrelin. Ghrelin significantly increased levels of proliferating cell nuclear antigen (PCNA) and cyclin D1, while reducing p27 in hepatocytes from mice fed NCD or HFD. Deletion of GHS-R1a completely abolished the effects of ghrelin in cultured hepatocytes. Ghrelin stimulated the phosphorylation of glycogen synthase kinase 3 beta (GSK3β), leading to subsequent increase of nuclear β-catenin in hepatocytes derived from lean and obese mice. This effect was dependent on the GHS-R1a. Conclusion: Ghrelin activates GHS-R1a to stimulate hepatocyte proliferation via GSK3/β-catenin signaling pathway.

Generation of a PXR reporter human induced pluripotent stem cell line (PXR-mCherry hiPSC) using the CRISPR/Cas9 system

Pregnane X receptor (PXR) is a key nuclear receptor that mediates drug metabolism and stimulates hepatocyte proliferation. However, the lack of PXR expression in human pluripotent stem cell-derived hepatocytes limits their application for drug screening and toxicity testing. Here, we generated a PXR-mCherry reporter human induced pluripotent stem cell (hiPSC) line using the CRISPR/Cas9 system. PXR-mCherry hiPSCs were pluripotent and had differentiation potential and a normal karyotype. This cell line is an important tool for identifying factors that increase PXR-mediated drug metabolism and hepatocyte proliferation.

Platelets in liver regeneration

Background and ObjectivesLoss of liver tissue leading to impairment of liver function represents a major cause of mortality. Understanding the mechanism of liver regeneration and developing therapies to sustain liver regeneration are of high therapeutic relevance. In this regard, platelets are considered as potential candidates for stimulating liver regeneration.MethodsWe aim to review the most recent evidence regarding the role of platelets in liver regeneration.ResultsPlatelets stimulate liver regeneration in animal models of liver resection. In humans, platelets are independent predictors of postoperative mortality, liver function and volume recovery. One proposed mechanism by which platelets stimulate liver regeneration relies on their direct effect on hepatocytes. Following partial hepatectomy, platelets accumulate in the residual liver and release their granule content. Platelet‐containing molecules, such as HGF, VEGF, IGF‐1 and serotonin, stimulate hepatocyte proliferation. A putative additional mechanism involves the transfer of platelet mRNA to hepatocytes following platelet internalization. Recent studies have suggested that the effect of platelets relies on their interactions with LSEC. Platelets induce the secretion of IL‐6 from LSEC, a strong initiator of hepatocyte proliferation. Additionally, platelets convey molecules that may impact LSEC function and, by extension, liver regeneration. Platelets potentially interact with Kupffer cells, but the effect of that interaction on liver regeneration remains to be determined.ConclusionPlatelets stimulate liver regeneration. Several mechanisms seem to be involved, acting on the level of hepatocytes, LSEC and potentially Kupffer cells. Identification of the platelet‐molecule(s) involved may lead to targeted therapies for patients with impairment of liver function.

Role of exogenous interleukin 4 in the dynamics of acute liver injury

BackgroundMortality in acute liver failure is very high, and the only reliable treatment is transplantation. Interleukin 4 (IL4) has pleiotropic effects on a network of cells directing repair, regeneration, and fibrosis. Pretreatment with subcutaneous IL4 complexed with anti-IL4 antibody (IL4c) promotes repair of carbon tetrachloride-mediated murine liver injury by stimulating hepatocyte proliferation. We hypothesised that IL4c could be administered therapeutically and act by a variety of mechanisms beyond engagement of hepatocytes. MethodsC57Bl/6 mice were given carbon tetrachloride intraperitoneally to induce acute liver injury. Preinjury and postinjury dosing regimens of IL4c were compared. The role of IL4 receptor α-chain (IL4Rα) signalling in bone-marrow-derived cells was investigated using tissue-protected chimeras generated with wild type or IL4Rα−/– donor bone marrow. Liver was analysed by immunohistochemistry and fluorescence-activated cell sorting (FACS). FindingsWhen compared with vehicle, administration of IL4c after injury produced a significant reduction in mean alanine aminotransferase (242 U/L [61·9] vs 131 [30·8], p<0·0001) and necrotic cell area (11·6% [7·04] vs 0·36 [0·6], p<0·0001). Although necrosis was reduced, the necrotic area in IL4c-treated liver was not replaced with hepatocytes but with a cellular infiltrate characterised by high F4/80 expression. FACs analysis showed a reduced proportion and number of Ly6chi MHCIIlo monocytes and a concomitant increase in the number of Ly6clo MHCIIhi F4/80hi macrophages in injured livers treated with IL4c. This phenotypic change was dependent on IL4c signalling to Ly6chi MHCIIlo monocytes, since these cells exhibited developmental arrest when derived from IL4Rα-deficient bone marrow in chimeric mice. InterpretationWe demonstrate that IL4c can be administered therapeutically after carbon tetrachloride injury to improve indices of hepatic injury. Furthermore, IL4c treatment mediates a change in the monocyte–macrophage compartment which is consistent with a switch from a proinflammatory (Ly6chi) to a proreparative (Ly6clo) macrophage phenotype. We postulate that these Ly6clo macrophages mediate clearance of necrotic debris and facilitate hepatic regeneration. FundingWellcome Trust.

Rosmarinic acid stimulates liver regeneration through the mTOR pathway

Background: Rosemary (Rosmarinus offcinsalis L) has a liver protection function under various conditions of liver damage. Rosmarinic acid, one of the pharmacological constituents of rosemary, exhibited protective effects against organ injury, including acute liver injury.Hypothesis: We hypothesize that RA stimulates liver regeneration.Study Design: In the present study, we investigated the effects and mechanism of RA administration on liver regeneration using partial hepatectomy (PH), a well-validated liver regeneration model in mice.Methods: We use a 2/3 partial hepatectomy (PH) model to induce liver regeneration. RA was administered prior to and simultaneously with PH. The regeneration process was estimated by the index of the liver to body weight (ILBW) and the expression of proliferating cell nuclear antigen (PCNA) and liver transaminases.Results: The administration of rosmarinic acid stimulated hepatocyte proliferation based on activation of the mTOR/S6K pathway. Rosmarinic acid treatment also rescued impaired liver function due to PH.Conclusion: These data demonstrate that RA is potentially useful to promote liver regeneration.

Mesenchymal stem cells and their secreted molecules predominantly ameliorate fulminant hepatic failure and chronic liver fibrosis in mice respectively.

BackgroundOrthotopic liver transplantation is the only effective treatment for liver failure but limited with shortage of available donor organs. Recent studies show promising results of mesenchymal stem cells (MSCs)-based therapies.MethodsWe systematically investigate the therapeutic effects of MSCs or MSC-conditioned medium (MSC-CM) in ameliorating fulminant hepatic failure (FHF) and chronic liver fibrosis in mice. In addition, extensive flow cytometry analysis of spleens from vehicle and MSC- and MSC-CM-treated mice was applied to reveal the alteration of inflammatory state.ResultsIn FHF model, MSCs treatment reduced remarkably the death incidents; the analysis of gross histopathology showed that control livers were soft and shrunken with extensive extravasated blood, which was gradually reduced at later time points, while MSC–treated livers showed gross pathological changes, even 24 h after MSC infusion, and hematoxylin and eosin staining revealed dramatical hepatocellular death with cytoplasmic vacuolization suppressed by MSCs treatment; flow cytometry analysis of total lymphocytes showed that macrophages (F4/80) infiltrated into control livers more than MSC-treated livers; by contrast, MSC-CM partially ameliorates FHF. In chronic liver injury model, MSC and MSC-CM both suppressed fibrogenesis and necroinflammatory, and the later was better; activation of hepatic stellate cells (α-SMA) was inhibited; glycogen synthesis and storage (indicated by periodic acid-Schiff -staining) was improved; liver regeneration (Ki67) was promoted while liver apoptosis (TUNEL) was reduced. In the in vitro, MSCs promote macrophage line RAW264.7 apoptosis and MSC-CM promotes apoptosis and inhibits proliferation of HSC line LX-2. We also found that MSCs and MSC-CM could improve spleen; MSC-CM increased levels of Th2 and Treg cells, and reduced levels of Th17 cells, whereas levels of Th1 cells were unchanged; comparatively, MSC treatment did not affect Th17 and Treg cells and only slightly alters inflammatory state; MSC and MSC-CM treatment both substantially down-regulated macrophages in the spleens.ConclusionBoth MSCs and MSC-CM exert therapeutic effects by acting on various key cells during the pathogenesis of FHF and chronic fibrosis, stimulating hepatocyte proliferation and suppressing apoptosis, down-regulating infiltrating macrophages, converting CD4+ T lymphocyte system into an anti-inflammatory state, and facilitating hepatic stellate cell death.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0792-1) contains supplementary material, which is available to authorized users.

NMR metabolic profiling of the liver following administrationof alcohol andthemushroom Ganoderma lucidum in rats

We have evaluated the efficiency of a metabonomic approach to metabolic phenotyping and detection of early metabolic changes under a toxic influence. For this purpose, a metabolic profiling of rat liver was performed with 1H NMR spectroscopy. Rat tissues from animals in three groups were analyzed. Group C consisted of control animals; animals in group A received alcohol repeatedly (15 % ethanol); and animals in group A+ R received alcohol in combination with a hepatoprotective herbal medicine (Reishi, Ganoderma lucidum) repeatedly. Noteworthy, alcohol consumption did not cause pathological changes, but stimulated hepatocyte proliferation. Our data suggest that changes in metabolite concentrations in A represent a typical metabolic response to alcohol consumption, namely decrease in glycine, leucine, isoleucine, valine, choline and lactate content, and increase in TMAO content. Treatment with Reishi (A+ R) had positive effects, in that it restored the levels of glycine, valine and TMAO. Furthermore, increase in NAD, ATP, UTP, succinate, pyranose, and acetate concentrations was observed in A+ R. A correlation was found between the valine, isoleucine, lactate, cho­line, and pyranose content and the num­ber of binuclear hepatocytes. Binuclear hepatocytes indicate proliferative activity, and the concentration of the metabolites participating in the formation of new hepatic cells decreases. Thus, the study of liver tissues by 1H NMR spectroscopy allows for detection of early changes in metabolite concentra­tions following chronic consumption of alcohol at insignificant doses. Consequently, 1H NMR spectro­scopy can serve as a promising approach to detecting alcohol-related liver pathologies and assessing the efficiency of the therapy used.