Proliferation Rate Of Hepatocytes Research Articles

Rich dynamics of a hepatitis C virus infection model with logistic proliferation and time delays

AbstractIn this paper, we study a dynamic model of hepatitis C virus (HCV) infection with density‐dependent proliferation of uninfected and infected hepatocytes and two time delays, which is derived from a three‐dimensional model by the quasi‐steady‐state approximation. The model can exhibit forward bifurcation or backward bifurcation, and an explicit control threshold parameter is obtained for the case of backward bifurcation. It is shown that if the proliferation rate of infected hepatocytes is greater than the proliferation rate of uninfected hepatocytes by a certain amount, it becomes more difficult for the virus to be removed. The model has rich dynamical properties: (i) In some parameter regions, bistability can occur; (ii) both time delays (virus‐to‐cell delay) and (cell‐to‐cell delay) can lead to Hopf bifurcations; (iii) same length of time delays and can lead to at most one stability switch, but different time delays can lead to multiple stability switches. Several sufficient conditions for the global stability of the disease‐free equilibrium and the endemic equilibrium are obtained for both forward and backward bifurcation scenarios. In particular, several sharp results on global stability are obtained. Theoretical and numerical results portray the complexity of viral evolutionary dynamics in chronic HCV‐infected patients.

Mettl14 mutation restrains liver regeneration by attenuating mitogens derived from non-parenchymal liver cells

Liver regeneration is a well-known systemic homeostatic phenomenon. The N6-methyladenosine (m6A) modification pathway has been associated with liver regeneration and hepatocellular carcinoma. m6A methyltransferases, such as methyltransferase 3 (METTL3) and methyltransferase 14 (METTL14), are involved in the hepatocyte-specific-regenerative pathway. To illustrate the role of METTL14, secreted from non-parenchymal liver cells, in the initiation phase of liver regeneration, we performed 70% partial hepatectomy (PH) in Mettl14 heterozygous (HET) and wild-type (WT) mice. Next, we analyzed the ratio of liver weight to body weight and the expression of mitogenic stimulators derived from non-parenchymal liver cells. Furthermore, we evaluated the expression of cell cycle-related genes and the hepatocyte proliferation rate via MKI67-immunostaining. During regeneration after PH, the weight ratio was lower in Mettl14 HET mice compared to WT mice. The expressions of hepatocyte growth factor (HGF) and tumor necrosis factor (TNF)-α, mitogens derived from non-parenchymal liver cells that stimulate the cell cycle, as well as the expressions of cyclin B1 and D1, which regulate the cell cycle, and the number of MKI67-positive cells, which indicate proliferative hepatocyte in the late G1-M phase, were significantly reduced in Mettl14 HET mice 72 h after PH. Our findings demonstrate that global Mettl14 mutation may interrupt the homeostasis of liver regeneration after an acute injury like PH by restraining certain mitogens, such as HGF and TNF-α, derived from sinusoidal endothelial cells, stellate cells, and Kupffer cells. These results provide new insights into the role of METTL14 in the clinical treatment strategies of liver disease. [BMB Reports 2022; 55(12): 633-638].

The effect of aging on the repeated-dose liver micronucleus assay using diethylnitrosamine

BackgroundThe repeated-dose liver micronucleus (RDLMN) assay has been well-developed and applied because of its simplicity and the ease of integration into general toxicity studies which is the preferred method from the 3R’s point of view. In this assay, we observed micronucleated hepatocytes which accumulated during a rather long-term dosing period. When considering integration into general toxicity studies, the effects of age of the animals used in the micronucleus assay becomes a major issue. The effect of age on the micronucleus induction rate has been reported in bone marrow micronucleus assays, and it is considered that the decrease in cell proliferation rate due to aging is the cause of the decrease in sensitivity. A decrease in sensitivity due to aging was also reported in a liver micronucleus assay using clofibrate and the cause is considered to be a decrease in hepatocyte proliferation activity due to aging. However, no actual decrease in hepatocyte proliferation rate due to aging has been reported. In addition, there are no reports, so far, on whether similar effects of aging appear when other substances were administered. To investigate the effects of aging in the RDLMN assay, this study focused on the effects of 14-day repeated administration of DEN, a well-known genotoxic hepatocarcinogen with the hepatocyte toxicity which should cause an elevation of cell proliferation rate as a reflective regeneration.ResultsThe liver micronuclei induced by DEN were equivalent between the two age groups (i.e., six and eight weeks of age at the start of dosing). In the histopathological examination for the liver, single cell necrosis, karyomegaly, and increased mitosis were observed in the hepatocytes, and the frequency and severity were increased dose-dependently. Ki-67 immunohistochemical analysis which can detect all cells in the cell cycle other than those in the G0 phase revealed dose-dependent increase of cell proliferation activity, and the difference between ages was not observed.ConclusionThe effect of aging on the RDLMN assay could not be recognized when DEN was administered for 14 days in rats. Meanwhile, it was supported by the histopathological examination and Ki-67 immunohistochemical analysis that such an effect of aging was masked by the compensatory hepatocyte proliferation which was induced by the hepatocyte toxicity of DEN.

In vitro effects of Eucommia ulmoides and its active components on the growth, lipid metabolism and collagen metabolism of grass carp (Ctenopharyngodon idellus) hepatocyte and intramuscular fibroblast.

Two experiments were conducted to investigate the in vitro effects of Eucommia ulmoides (E. ulmoides) and its active components on the growth, lipid metabolism and collagen metabolism of grass carp's (Ctenopharyngodon idellus) hepatocytes and intramuscular fibroblasts. In experiments 1 and 2 (Expt. 1, 2), hepatocytes and intramuscular fibroblasts were treated with 2.5, 5, 10, 20, 40 and 80 μg ml-1 of Eucommia bark extract (EBE), Eucommia leaf extract (ELE), pinoresinol diglucoside (PDG), chlorogenic acid (CGA), quercetin (QC) and aucubin (AU) for 24 h, respectively, then the cell growth, lipid and collagen metabolism-related gene expressions were evaluated. The results showed that the cell proliferation rate of hepatocytes and intramuscular fibroblasts was significantly improved by the supplementation of EBE, ELE, CGA, QC and AU. Moreover, triglyceride concentration of hepatocytes was significantly decreased by the EBE, ELE, CGA and QC supplementations compared to the control. Meanwhile, EBE, ELE, CGA, QC and AU supplementations significantly upregulated the relative gene expressions of insulin-like growth factor-1 (igf1), protein kinase B (akt), target of rapamycin (tor) and eukaryotic initiation factor 4E binding protein 1 (4ebp1) in hepatocytes, and ribosomal protein S6 kinase 1 (s6k1) transcription was significantly activated by ELE, CGA and QC supplementations. Nonetheless, phosphatidylinositol 3-kinase (pi3k) was unaffected by any of the supplements. In addition, the mRNA expressions of genes associated with lipid metabolism (peroxisome proliferator activated receptor α pparα, carnitine palmitoyltransferase 1 cpt1, adipose triglyceride lipase atgl, hormone-sensitive lipase hsl, peroxisome proliferator activated receptor γ pparγ) were significantly upregulated by EBE, ELE, CGA and QC. In intramuscular fibroblasts, the EBE, ELE, CGA, QC and AU supplementations significantly increased in vitro hydroxyproline concentrations, promoted the relative expressions of transforming growth factor-β1 (tgfβ1), connective tissue growth factor (ctgf), collagen type I alpha 1/2 chain (col1a1, col1a2), lysine oxidase (lox) and tissue inhibitor of matrix metalloproteinase-2 (timp2), and decreased matrix metalloproteinase-2 (mmp2) gene expression. Also, the gene expressions of drosophila mothers against decapentaplegic protein 2/4 (smad2, smad4) and proline hydroxylase (phd) were significantly upregulated by ELE, CGA, QC and AU supplementations. Based on the present in vitro results of grass carp, EBE, ELE, CGA, QC and AU improved the growth and lipid metabolism (except AU) in hepatocytes, and promoted the collagen deposition in intramuscular fibroblast, which is partly attributed to the signalling pathways of AKT/TOR, PPARα and TGF-β/Smads/CTGF.

Size of portally deprived liver lobe after portal vein ligation and additional partial hepatectomy: Result of balancing proliferation and apoptosis

The liver has the ability to maintain its total size by adjusting the size of the individual liver lobes differently in response to regeneration- and atrophy-stimuli. Portal vein ligation (PVL) drives the ligated lobe to undergo atrophy whereas partial hepatectomy (PHx) drives the total remnant liver to regenerate. We hypothesize that the size of the PVL-lobe is dependent on the balance between the extent of PVL and the extent of PHx inducing a complex interplay between hepatocyte proliferation, apoptosis and autophagy. Lewis-rats were subjected to either 20%PVL + 70%PHx or 70%PVL + 20%PHx. Control groups consisted of 20%PVL and 70%PVL. Liver lobe weight, BrdU-proliferation-index, proliferating-cell-nuclear-antigen-mRNA-expression level, apoptotic density and autophagy-related-proteins were investigated. The PVL-liver lobe adjusted its weight differently, increasing by 40% after 20%PVL + 70%PHx, but decreasing by 25% after 70%PVL + 20%PHx. Additional resection induced a low, but substantial size-dependent hepatocyte proliferation rate (maximal 6.3% and 3.6% vs. 0.3% and significantly suppressed apoptotic density in the deportalized-liver-lobe (3 and 14 cells/mm2 comparing with above 26 cells/mm2, p < 0.01). Autophagy was more activated in PVL-liver lobe after simultaneous PHx than after PVL only. In summary, atrophy of the PVL-liver lobe after simultaneous PHx was counteracted by promoting hepatocyte proliferation, inducing autophagy and suppressing apoptosis in a PHx-extent-dependent manner.

Sensitivity analysis of chronic hepatitis C virus infection with immune response and cell proliferation

A new mathematical model of chronic hepatitis C virus (HCV) infection incorporating humoral and cell-mediated immune responses, distinct cell proliferation rates for both uninfected and infected hepatocytes, and antiviral treatment all at once, is formulated and analyzed meticulously. Analysis of the model elucidates the existence of multiple equilibrium states. Moreover, the model has a locally asymptotically stable disease-free equilibrium (DFE) whenever the basic reproduction number is less than unity. Local sensitivity analysis (LSA) result exhibits that the most influential (negatively sensitive) parameters on the epidemic threshold are the drug efficacy of blocking virus production and the drug efficacy of removing infection. However, LSA does not accurately assess uncertainty and sensitivity in the system and may mislead us since by default this technique holds all other parameters fixed at baseline values. To overcome this pitfall, one of the most robust and efficient global sensitivity analysis (GSA) methods, which is Latin hypercube sampling-partial rank correlation coefficient technique, elucidates that the proliferation rate of infected hepatocytes and the drug efficacy of killing infected hepatocytes are highly sensitive parameters that affect the transmission dynamics of HCV in any population. Our study suggests that cell proliferation of the infected hepatocytes can be very crucial in controlling disease outbreak. Thus, a future HCV drug that boosts cell-mediated immune response is expected to be quite effective in controlling disease outbreak.

Cyclin-like proteins tip regenerative balance in the liver to favour cancer formation.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. A variety of factors can contribute to the onset of this disease, including viral infection, obesity, alcohol abuse and non-alcoholic fatty liver disease (NAFLD). These stressors predominantly introduce chronic inflammation leading to liver cirrhosis and finally the onset of HCC; however, approximately 20% of HCC cases arise in the absence of cirrhosis via a poorly defined mechanism. The atypical cyclin-like protein Spy1 is capable of overriding cell cycle checkpoints, promoting proliferation and has been implicated in HCC. We hypothesize that Spy1 promotes sustained proliferation making the liver more susceptible to accumulation of deleterious mutations, leading to the development of non-cirrhotic HCC. We report for the first time that elevation of Spy1 within the liver of a transgenic mouse model leads to enhanced spontaneous liver tumourigenesis. We show that the abundance of Spy1 enhanced fat deposition within the liver and decreased the inflammatory response. Interestingly, Spy1 transgenic mice have a significant reduction in fibrosis and sustained rates of hepatocyte proliferation, and endogenous levels of Spy1 are downregulated during the normal fibrotic response. Our results provide support that abnormal regulation of Spy1 protein drives liver tumorigenesis in the absence of elevated fibrosis and, hence, may represent a potential mechanism behind non-cirrhotic HCC. This work may implicate Spy1 as a prognostic indicator and/or potential target in the treatment of diseases of the liver, such as HCC. The cyclin-like protein Spy1 enhances lipid deposition and reduces fibrosis in the liver. Spy1 also promotes increased hepatocyte proliferation and onset of non-cirrhotic hepatocellular carcinoma (HCC). Thus, Spy1 may be used as a potential target in the treatment of HCC.

A preliminary study of associating liver partition and portal vein ligation for staged hepatectomy in a rat model of liver cirrhosis.

Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) in a rat model of liver cirrhosis has not, to the best of our knowledge, been previously investigated. The present study therefore aimed to establish a model of ALPPS in cirrhotic rats and to assess liver regeneration. Rats were randomly divided into an ALPPS group with carbon tetrachloride-induced cirrhosis (group A) and a normal liver (group B). Rat weight, cytokine levels, biochemical parameters and histopathology were assessed 1, 2, 3, 7 and 14 days after ALPPS. Higher aspartate aminotransferase and alanine aminotransferase levels were detected in group A on the first postoperative day. On the first, second and third days, hepatocyte proliferation rate was higher in group B than in group A. After 3 days, hepatocyte proliferation rate in group B began to decrease, but the rate in group A continued to increase until the 14th day. Higher levels of hepatocyte growth factor, interleukin-6 and tumor necrosis factor-α were detected in group A compared with group B, but the differences were not significant. The present study demonstrated that ALPPS promoted liver regeneration in a rat model of cirrhosis, but significantly impaired liver function. Compared with the ALPPS model, group B exhibited a delayed peak of proliferation. The mechanism of liver regeneration induced by ALPPS in cirrhotic rats may be associated with increased cytokine levels.

Immunotherapy of tacrolimus in small volume liver transplantation of rats

Objective To detect the plasma concentration of tacrolimus and the survival time of rats after small-volume liver transplantation, and to investigate the criteria for immunological rejection after small-volume liver transplantation. Methods Lewis rats and Brown Norway rats were used to establish a small volume and normal liver volume transplantation model, which were divided into 7 groups: whole liver transplantation group (WI), small volume allogeneic liver transplantation group (SI), and whole liver allograft group (WA), small volume allogeneic liver transplantation group (SA), whole liver allograft immunotherapy group (WAT), small volume allogeneic liver transplantation immunotherapy group (SAT), small volume allogeneic liver transplantation immunotherapy modulation group (SATa). Morphological and functional changes of liver tissue were studied postoperatively, AST and tacrolimus plasma concentrations were detected, and survival was recorded. Results Compared with the WA group, the inflammatory cells infiltrated in the portal area of the SA group, the inflammatory changes of the sinusoidal endothelial cells, and the proportion of TCRpositive lymphocytes increased. Four days after transplantation, peripheral blood tests showed that CD4+ CD25+ double positive lymphocytes were significantly lower in the allograft group than in the allograft group, and the positive expression rate in the SA group (0.6%) was significantly lower than that in the WA group (1.8%). The differences were statistically significant (P<0.05). In the SAT group, the blood concentration of tacrolimus was significantly higher than that in the WAT group at each time point (P<0.05). The blood concentration of tacrolimus in the SATa group was relatively stable, and the plasma concentration of the SATa group was stable. And AST was significantly lower than the SAT group, the differences were statistically significant (P<0.05). Compared with WAT group, the proliferation and apoptosis rate of hepatocytes in SAT group and SATa group were significantly increased. The proliferation of hepatocytes in SATa group was significantly higher than that in SAT group (P<0.05). Survival analysis showed that the cumulative survival rate of the WA group was 85.7%, which was significantly higher than that of the SAT group (28.6%). The difference was statistically significant (P<0.05). The cumulative survival rate of the SATa group was 51.7%. The survival time of WAt group was (57.4±25.0) days, SAT group was (28.0±29.10) days, SATa group was (39.7±29.0) days, which were longer than untreated groups. The ratio of proliferation to apoptosis (PRA) increased with increasing time of tacrolimus. Regardless of blood concentration, tacrolimus plasma concentration was positively correlated with AST (R=0.758, P<0.05), indicating RPA was inversely correlated with AST (R=-0.962, P<0.05). Conclusion The use of tacrolimus significantly prolonged the survival time of small-volume allogeneic liver transplantation rats. Adjusting the amount of tacrolimus under the guidance of tacrolimus plasma concentration and AST serum value equation TD=-0.494TC-0.0035AST+ 260.487 to make the blood concentration relatively stable, it can further extend the allogeneic liver transplantation rat time to live. Key words: Small volume liver transplantation; Tacrolimus; Immune rejection

Multipotent stromal cells stimulate liver regeneration by influencing the macrophage polarization in rat

AIMTo investigate the influence of the umbilical cord-derived multipotent stromal cells (MSCs) on recovery of the liver after the subtotal resection, that is, removal of 80% of the organ mass, a renowned model of the small-for-size liver remnant syndrome.METHODSThe MSCs were obtained from the intervascular tissue of umbilical cords, dissected from rat fetuses, by the explant culture technique. The vital labeling of MSCs with РКН26 was carried out on the 3rd passage. The subtotal resection was performed on male Sprague-Dawley rats. The experimental group animals received a transplant 106 MSCs infused into the spleen. Hepatocyte proliferation was assessed by counting of either mitotic figures or Ki67-positive cells in microscopic images. MSC differentiation was assessed with antibodies to hepatocyte-specific marker cytokeratin 18 (CK18), cholangiocyte-specific protein CK19, smooth muscle cell-specific protein α-SMA, the endothelial cell marker CD31, or the active fibroblast marker FAPα. Total macrophages of the liver were selectively stained in cryosections incubated with anti-CD68 antibodies (1:100, Abcam), while the M2a and M2c macrophage populations were selectively stained with anti-CD206 antibodies. Expression of interleukin and growth factor genes was evaluated with PCR-RT.RESULTSIntrasplenic allogeneic transplantation of the umbilical cord-derived multipotent stromal cells stimulates reparative processes within the residual liver tissue after subtotal resection (removal of 80% of the organ mass), as indicated by increased rates of hepatocyte proliferation and accelerated organ mass recovery. These effects may result from paracrine influence of the transplanted cells on the resident macrophage population of the liver. The transplantation favors polarization of macrophages to M2 phenotype (the M2-polarized macrophages specifically express CD206; they are known to suppress inflammation and support tissue repair). No differentiation of the transplanted cells into any of the liver cell types have been observed in the study.CONCLUSIONWe found no direct evidence for the paracrine effect of MSCs on liver regeneration after the subtotal liver resection in rats. However, the paracrine mechanism of the therapeutic activity of transplanted MSC is indirectly indicated by a decrease in the total number of CD68 + macrophages and an increase in the proportion of M2 pro-repair macrophages in the regenerating liver as compared to animals in which the transplantation was only mimicked.

Role of CYP2B in Phenobarbital-Induced Hepatocyte Proliferation in Mice.

Phenobarbital (PB) promotes liver tumorigenesis in rodents, in part through activation of the constitutive androstane receptor (CAR) and the consequent changes in hepatic gene expression and increases in hepatocyte proliferation. A typical effect of CAR activation by PB is a marked induction of Cyp2b10 expression in the liver; the latter has been suspected to be vital for PB-induced hepatocellular proliferation. This hypothesis was tested here by using a Cyp2a(4/5)bgs-null (null) mouse model in which all Cyp2b genes are deleted. Adult male and female wild-type (WT) and null mice were treated intraperitoneally with PB at 50 mg/kg once daily for 5 successive days and tested on day 6. The liver-to-body weight ratio, an indicator of liver hypertrophy, was increased by 47% in male WT mice, but by only 22% in male Cyp2a(4/5)bgs-null mice, by the PB treatment. The fractions of bromodeoxyuridine-positive hepatocyte nuclei, assessed as a measure of the rate of hepatocyte proliferation, were also significantly lower in PB-treated male null mice compared with PB-treated male WT mice. However, whereas few proliferating hepatocytes were detected in saline-treated mice, many proliferating hepatocytes were still detected in PB-treated male null mice. In contrast, female WT mice were much less sensitive than male WT mice to PB-induced hepatocyte proliferation, and PB-treated female WT and PB-treated female null mice did not show significant difference in rates of hepatocyte proliferation. These results indicate that CYP2B induction plays a significant, but partial, role in PB-induced hepatocyte proliferation in male mice.

Dicer-dependent production of microRNA221 in hepatocytes inhibits p27 and is required for liver regeneration in mice.

Dicer processes microRNAs (miRs) into active forms in a wide variety of tissues, including the liver. To determine the role of Dicer in liver regeneration, we performed a series of in vivo and in vitro studies in a murine 2/3 hepatectomy model. Dicer was downregulated after 2/3 hepatectomy, and loss of Dicer inhibited liver regeneration associated with decreased cyclin A2 and miR-221, as well as increased levels of the cell cycle inhibitor p27. In vitro, miR-221 inhibited p27 production in primary hepatocytes and increased hepatocyte proliferation. Specific reconstitution of miR-221 in hepatocyte-specific Dicer-null mice inhibited p27 and restored liver regeneration. In wild type mice, targeted inhibition of miR-221 using a cholesterol-conjugated miR-221 inhibited hepatocyte proliferation after 2/3 hepatectomy. These results identify Dicer production of miR-221 as an essential component of a miRNA-dependent mechanism for suppression of p27 that controls the rate of hepatocyte proliferation after partial hepatectomy.NEW & NOTEWORTHY Our findings demonstrate a direct role for microRNAs in controlling the rate of liver regeneration after injury. By deleting Dicer, an enzyme responsible for processing microRNAs into mature forms, we determined miR-221 is a critical microRNA in the physiological process of restoration of liver mass after injury. miR-221 suppresses p27, releasing its inhibitory effects on hepatocyte proliferation. Pharmaceuticals based on miR-221 may be useful to modulate hepatocyte proliferation in the setting of liver injury.

Denudation of human amniotic membrane by a novel process and its characterisations for biomedical applications.

This study was aimed to investigate the suitability of a modified method to get decellularised human amniotic membrane (DHAM). The obtained membrane was subjected to physico-chemical and biological evaluations to validate its potential for biomedical applications. The human amniotic membrane was processed with detergent and alkali followed by enzymatic treatments. Hematoxylin and eosin (H&E) and Masson’s trichrome staining of membrane were in accordance with conjectures: the decellularised membrane stained for extracellular matrix is rich in collagen. Scanning electron micrograph also showed the denudation in the processed membrane with the cellular impressions on the basement membrane. Physical characteristics namely the differential scanning calorimetric, tensile, shrinkage behaviour and the Fourier transform infrared spectra of decellularised membrane showed its stability and intact structure similar to the unprocessed membrane. In the visible range of light, the membrane was found to be transparent from 90 to 98 %. Proliferation rate of fibroblasts, keratinocytes, myoblasts and hepatocytes were significantly upregulated compared to the control. The cell morphologies were normal and differentiation of myoblasts into myotubes were more pronounced in decellularised membrane. Proliferation of corneal limbal cells on decellularised membrane showed 92–100 % confluency on day 21 and the migrated cells displayed a spindle shape and changing later to a more cuboidal appearance.

SOCS2 Balances Metabolic and Restorative Requirements during Liver Regeneration

After significant injury, the liver must maintain homeostasis during the regenerative process. We hypothesized the existence of mechanisms to limit hepatocyte proliferation after injury to maintain metabolic and synthetic function. A screen for candidates revealed suppressor of cytokine signaling 2 (SOCS2), an inhibitor of growth hormone (GH) signaling, was strongly induced after partial hepatectomy. Using genetic deletion and administration of various factors we investigated the role of SOCS2 during liver regeneration. SOCS2 preserves liver function by restraining the first round of hepatocyte proliferation after partial hepatectomy by preventing increases in growth hormone receptor (GHR) via ubiquitination, suppressing GH pathway activity. At later times, SOCS2 enhances hepatocyte proliferation by modulating a decrease in serum insulin-like growth factor 1 (IGF-1) that allows GH release from the pituitary. SOCS2, therefore, plays a dual role in modulating the rate of hepatocyte proliferation. In particular, this is the first demonstration of an endogenous mechanism to limit hepatocyte proliferation after injury.

Evaluation of repeated dose micronucleus assays of the liver using N-nitrosopyrrolidine: A report of the collaborative study by CSGMT/JEMS.MMS

The repeated dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens, and can be integrated into a general toxicological study. To assess the performance of the assay, N-nitrosopyrrolidine (NPYR), a genotoxic hepatocarcinogen, was tested in 14- or 28-day RDLMN assays. NPYR was orally administered to rats at a daily dose of 25, 50 or 100mg/kg. One day after the last administration, a portion of the liver was removed and hepatocyte micronucleus (MN) specimens were prepared by the new method recently established by Narumi et al. In addition, a bone marrow MN assay and a histopathological examination of the liver were conducted. The detection of Phospho-Histone H3 was performed by immunohistochemistry to evaluate the proliferation rate of hepatocytes. The results showed significant increase in the number of micronucleated hepatocytes and Phospho-Histone H3-positive cells from the lowest dose in both 14- and 28-day RDLMN assays. On the other hand, the bone marrow MN assay yielded a negative result, which was in accordance with the existing report of the bone marrow MN assay using mice. Upon histopathological examination, inflammatory lesions and hypertrophy were noted, which may explain the increase in the hepatocyte proliferation and the enhancement of MN induction by NPYR. Our findings indicate that the RDLMN assay could be a useful tool for comprehensive risk assessment of carcinogenicity by providing information on both genotoxicity and histopathology when integrated into a general repeat dosing toxicity assay.

Pretreatment with recombinant human interleukin 2 (rhIL-2) Up-regulates PCNA-positive cells after partial hepatectomy in rat liver

We prepared recombinant human interleukin-2 (rhIL-2) and studied its pretreated influence on liver regeneration and the blood profile in partially (67%) hepatectomized (PH) male Sprague-Dawley rats. Rats were injected in the tail vein with rhIL-2 three times per day for 3 consecutive days and 67% underwent a partial hepatectomy (PH). Five days after the PH, liver tissue and blood samples were analyzed for liver regeneration and hematological changes. The weight of the liver in the rhIL-2 pretreated groups increased in a dose-dependent manner; with the highest treatment (24 × 104 IU/kg), the maximum liver weight of 88.6% was exhibited. The control group showed a gradual increase to 76.3% of the original liver weight. A histological analysis of the liver showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells in rhIL-2 pretreated rat livers. The rate of hepatocyte proliferation also increased significantly in primary cultured rat liver cells following rhIL-2 treatment. These results suggest that pretreatment with rhIL-2 may play adjuvant roles in liver regeneration after PH.

Interleukin 18 accelerates the hepatic cell proliferation in rat liver regeneration after partial hepatectomy

Interleukin 18 (IL-18) is a proinflammatory cytokine with an ability to accelerate cell proliferation through activating other factors. However, little is yet understood of the role of IL-18 in the regulation of liver regeneration (LR). To study the effect of IL-18 on LR, the gene expression profiles of hepatocytes isolated from rat regenerative liver were determined by Rat Genome 230 2.0 microarray. Next, the synergistic effects of genes associated to IL-18 pathway were analyzed by expression profile function Et. Then, the expression level of IL-18 was examined by RT-PCR and ELISA. Finally, the effect of IL-18 on hepatocyte proliferation was detected by injecting recombinant rat IL-18 (rrIL-18) into rats immediately after partial hepatectomy (PH) and the rate of hepatocyte proliferation was detected by BrdU labeling. The microarray result showed that the expressions of 13 genes of IL-18 pathway and 49 cell proliferation genes regulated by the pathway were significantly altered at transcriptional level. The Et values of three branches of IL-18 pathway, NF-κB, p38 and JNK, were markedly enhanced during the priming and progressing phases of rat LR. The mRNA level of IL-18 was significantly elevated at 2 and 36h, and its level in plasma was also significantly increased at 2h, and reached the peaks at 12h and 48h after PH (p<0.05). The number of BrdU positive cells was dramatically increased in rats treated with IL-18 compared to PBS control group (p<0.01). In conclusion, IL-18 promotes rat hepatocyte proliferation in the LR priming and progressing phases after PH.

CYLD controls c-MYC expression through the JNK-dependent signaling pathway in hepatocellular carcinoma

Posttranslational modification of different proteins via direct ubiquitin attachment is vital for mediating various cellular processes. Cylindromatosis (CYLD), a deubiquitination enzyme, is able to cleave the polyubiquitin chains from the substrate and to regulate different signaling pathways. Loss, or reduced expression, of CYLD is observed in different types of human cancer, such as hepatocellular carcinoma (HCC). However, the molecular mechanism by which CYLD affects cancerogenesis has to date not been unveiled. The aim of the present study was to examine how CYLD regulates cellular functions and signaling pathways during hepatocancerogenesis. We found that mice lacking CYLD were highly susceptible to chemically induced liver cancer. The mechanism behind proved to be an elevated proliferation rate of hepatocytes, owing to sustained c-Jun N-terminal kinase 1 (JNK1)-mediated signaling via ubiquitination of TNF receptor-associated factor 2 and expression of c-MYC. Overexpression of wild-type CYLD in HCC cell lines prevented cell proliferation, without affecting apoptosis, adhesion and migration. A combined immunohistochemical and tissue microarray analysis of 81 human HCC tissues revealed that CYLD expression is negatively correlated with expression of proliferation markers Ki-67 and c-MYC. To conclude, we found that downregulation of CYLD induces tumor cell proliferation, consequently contributing to the aggressive growth of HCC. Our findings suggest that CYLD holds potential to serve as a marker for HCC progression, and its link to c-MYC via JNK1 may provide the foundation for new therapeutic strategies for HCC patients.

Combined therapy of mesenchymal stem cell transplantation and interleukin 1 receptor antagonist for swine acute liver failure

Objective To evaluate synergic effects of allogeneic mesenchymal stem cells (MSCs) transplantation and interleukin 1 receptor antagonist (IL-1Ra) on swine acute liver failure.Methods Chinese experimental mini swine were given D-galactosamine to establish models of acute liver failure.Twentyone pigs were randomly divided into three groups (n =7 each).In the control group (A),the normal saline was injected into the liver via the portal vein after induction for 24 h; In the MSCs transplantation group (B),8 × 107 MSCs (in 40 ml PBS) were injected into the liver via the portal vein after induction for 24 h;In combined therapy group (C),8 × 107 MSCs were injected into the liver via the portal vein after induction for 24 h,followed by injection of rhIL-1Ra (10 mg/pig) via the ear vein at 6 h before,and one day and three days after transplantation respectively.Liver function,serum inflammation and pathological changes were measured.The fate of MSCs was also observed.Results The biochemical assay,the serum inflammation level and pathological changes in group C were all greatly different from those in group A and group B (P ＜ 0.05).The proliferation rate of hepatocytes in group C was 67.70 ± 9.47,71.80 ± 9.45,and 40.04 ±4.60 one week after transplantation,significantly higher than in group A and group B (P ＜ 0.05).The number of MSCs in group C was greater than in group B.Hepatocytes regeneration were associated with the kinesis changes of inflammation.Our findings suggest IL-1 Ra as an important player in the repair of acute liver failure (ALF).Improving homeostasis of liver failure contributed to increased hepatic engraftment and hepatocytes proliferation.Conclusion The MSCs transplantation is useful for ALF partially.The combined therapy shows cooperative effects. Key words: Interleukin 1 receptor antagonist ; Mesenchymal stem cells ; Cell transplantation ; Acute liver failure

Enhancement of Liver Regeneration by Adenosine Triphosphate-Sensitive K+ Channel Opener (Diazoxide) After Partial Hepatectomy

Enhancement of liver regeneration is a matter of importance after partial liver transplantation including small-for-size grafting. Mitochondrial adenosine triphosphate (ATP)-sensitive K⁺ (mitoKATP) channel plays an important role in mitochondrial bioenergetics, which is a prerequisite for liver regeneration. However, the ATP-sensitive K⁺ (KATP) channel in hepatocytes is incompletely understood. We investigated the KATP channel in hepatocytes and examined the effects of diazoxide, a potent KATP channel opener, on liver regeneration using a rat model. Using rat primary hepatocytes, expression and localization of KATP channel subunits, Kir6.x and sulfonylurea receptor (SUR)x, were studied by polymerase chain reaction, Western blotting, and immunostaining. To investigate the role of KATP channel openers in liver regeneration, we allocated rats into four groups: control (vehicle) (n=24), diazoxide (n=24), vehicle plus channel blocker (n=6), and diazoxide plus channel blocker (n=6) groups. After 70% partial hepatectomy, hepatic tissue ATP levels, liver-to-body weight ratio, and proliferation rate of hepatocytes were examined. KATP channel subunits, Kir6.1 and SUR1, were detected on hepatic mitochondria. During liver regeneration, liver-to-body weight ratio, proliferation rate of hepatocytes, and the hepatic ATP level were significantly higher in the diazoxide group than the control group at 2 days after partial hepatectomy. These effects of diazoxide were neutralized by a KATP channel blocker. We demonstrated the existence of a mitoKATP channel in hepatocytes composed of Kir6.1 and SUR1. Diazoxide could enhance liver regeneration by keeping a higher ATP content of the liver tissue. These results suggest that diazoxide will sustain the mitochondrial energetics through the mitoKATP channel opening.