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1.
J Cell Biochem ; 120(8): 13254-13261, 2019 08.
Article in English | MEDLINE | ID: mdl-30924172

ABSTRACT

To study the effect of miRNA-200b on hepatic fibrosis induced by CCl4 in mice. The C59BL/6 mice were randomly divided into three groups (normal control [NC], CCLR model [Model], and CCl 4 + miRNA-200b [miRNA]). The hepatic fibrosis was induced by CCl 4 injected subcutaneously twice per week in Model and miRNA groups. After 6 weeks building model, the mice of miRNA group were injected the miRNA-200b from caudal vein twice per week. The mice of Model and miRNA groups were continuously fed for 3 weeks. The IL-1ß, IL-6, and TNF-α concentrations of serum were measured by enzyme-linked immunosorbent assay. The hepatic tissues of difference groups were observed by hematoxylin and eosin (H&E) staining, sirius red staining, Masson staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay and measured toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) proteins expressions by western blot assay. The correlation between miRNA-200b and TLR4 were analyzed by dual luciferase target assay. Compared with NC group, the interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) concentrations of Model group were significantly upregulated (P < 0.05, respectively). With miRNA-200b overexpression, the IL-1ß, IL-6, and TNF-α concentrations were significantly suppressed (P < 0.05, respectively). The pathologies were improved by H&E staining, sirius red staining, and Masson staining; meanwhile, the hepatic cell apoptosis rate was significantly suppressed (P < 0.05). The TLR4 and NF-κB protein expressions of miRNA group were significantly suppressed compared with the Model group (P < 0.05, respectively). By dual luciferase target assay, the TLR4 was a target gene of miRNA-200b. The miRNA-200b upregulation improved hepatic fibrosis induced by CCl 4 via regulation of TLR4 in vivo.


Subject(s)
Chemokine CCL4/toxicity , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , MicroRNAs/therapeutic use , Toll-Like Receptor 4/metabolism , Animals , Enzyme-Linked Immunosorbent Assay , In Situ Nick-End Labeling , Liver Cirrhosis/chemically induced , Male , Mice
2.
Growth Factors ; 35(4-5): 144-160, 2017 10.
Article in English | MEDLINE | ID: mdl-29110545

ABSTRACT

Cellular therapies hold promise to alleviate liver diseases. This study explored the potential of allogenic serum isolated from rat with acute CCl4 injury to differentiate adipose derived stem cells (ADSCs) towards hepatic lineage. Acute liver injury was induced by CCl4 which caused significant increase in serum levels of VEGF, SDF1α and EGF. ADSCs were preconditioned with 3% serum isolated from normal and acute liver injury models. ADSCs showed enhanced expression of hepatic markers (AFP, albumin, CK8 and CK19). These differentiated ADSCs were transplanted intra-hepatically in CCl4-induced liver fibrosis model. After one month of transplantation, fibrosis and liver functions (alkaline phosphatase, ALAT and bilirubin) showed marked improvement in acute injury group. Elevated expression of hepatic (AFP, albumin, CK 18 and HNF4a) and pro survival markers (PCNA and VEGF) and improvement in liver architecture as deduced from results of alpha smooth muscle actin, Sirius red and Masson's trichome staining was observed.


Subject(s)
Cell Differentiation , Chemokine CCL4/blood , Culture Media, Conditioned/pharmacology , Intercellular Signaling Peptides and Proteins/pharmacology , Liver Cirrhosis/therapy , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/cytology , Adipose Tissue/cytology , Animals , Chemokine CCL4/toxicity , Hepatocytes/cytology , Hepatocytes/drug effects , Intercellular Signaling Peptides and Proteins/metabolism , Liver Cirrhosis/etiology , Mesenchymal Stem Cells/drug effects , Rats , Rats, Sprague-Dawley
3.
J Hepatol ; 63(2): 446-55, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25819337

ABSTRACT

BACKGROUND & AIMS: Large liver cell changes (LLCC) are characterized by pleomorphic large nuclei frequently found in liver diseases as chronic viral hepatitis and liver cirrhosis. The origin of this lesion remains cryptic, but the presence of LLCC is correlated with an increased risk of hepatocellular carcinoma. Telomeric repeat binding factor 1 (TRF1) is part of the shelterin complex and is essential for telomere protection. Ablation of TRF1 induces telomere fragility and fusions and chromosomal instability. METHODS: In this study, we addressed the role of TRF1 in liver regeneration generating a mouse model with conditional deletion of TRF1 in the liver. RESULTS: TRF1 deletion has no deleterious effects in liver and leads to increased ploidy of hepatocytes after 2/3 hepatectomy. Mice lacking TRF1 in the liver can survive for over one year without any evidence for altered liver function. Importantly, applying chronic replicative stress by frequent carbon tetrachloride (CCl4) injections, TRF1 deleted mice undergo ploidy changes consistent with endoreduplication and develop LLCC like lesions in the liver positive for p21, Cyclin D1 and PCNA as observed in humans. CONCLUSION: In summary, we provide mechanistic insight into the role of TRF1 in liver regeneration and provide a mouse model recapitulating the clinical features of LLCC.


Subject(s)
Carcinoma, Hepatocellular/genetics , Liver Neoplasms, Experimental/genetics , RNA, Neoplasm/genetics , Telomeric Repeat Binding Protein 1/genetics , Animals , Blotting, Western , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Chemokine CCL4/toxicity , Hepatocytes/metabolism , Immunohistochemistry , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Mice , Mice, Knockout , Real-Time Polymerase Chain Reaction
4.
Am J Physiol Gastrointest Liver Physiol ; 302(4): G420-9, 2012 Feb 15.
Article in English | MEDLINE | ID: mdl-22159278

ABSTRACT

Previous studies have demonstrated that mice disrupted with the cyclooxygenase-2 gene showed much more severe liver damage compared with wild-type mice after liver injury, and prostaglandins (PGs) such as PGE(1/2) and PGI(2) have decreased hepatic injury, but the mechanisms by which prostaglandins exhibit protective action on the liver have yet to be addressed. In the present study, we investigated the mechanism of the protective action of PGI(2) using the synthetic IP receptor agonist ONO-1301. In primary cultures of hepatocytes and nonparenchymal liver cells, ONO-1301 did not show protective action directly on hepatocytes, whereas it stimulated expression of hepatocyte growth factor (HGF) in nonparenchymal liver cells. In mice, peroral administration of ONO-1301 increased hepatic gene expression and protein levels of HGF. Injections of CCl4 induced acute liver injury in mice, but the onset of acute liver injury was strongly suppressed by administration of ONO-1301. The increases in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by CCl4 were suppressed by 10 mg/kg ONO-1301 to 39.4 and 33.6%, respectively. When neutralizing antibody against HGF was administered with ONO-1301 and CCl4, the decreases by ONO-1301 in serum ALT and AST, apoptotic liver cells, and expansion of necrotic areas in liver tissue were strongly reversed by neutralization of endogenous HGF. These results indicate that ONO-1301 increases expression of HGF and that hepatoprotective action of ONO-1301 in CCl4-induced liver injury may be attributable to its activity to induce expression of HGF, at least in part. The potential for involvement of HGF-Met-mediated signaling in the hepatotrophic action of endogenous prostaglandins generated by injury-dependent cyclooxygenase-2 induction is considerable.


Subject(s)
Chemical and Drug Induced Liver Injury/prevention & control , Chemokine CCL4/toxicity , Hepatocyte Growth Factor/metabolism , Hepatocytes/drug effects , Pyridines/pharmacology , Receptors, Prostaglandin/agonists , Alanine Transaminase/blood , Animals , Aspartate Aminotransferases/blood , Cells, Cultured , Chemical and Drug Induced Liver Injury/metabolism , Dose-Response Relationship, Drug , Female , Hepatocyte Growth Factor/genetics , Hepatocytes/metabolism , Liver/drug effects , Liver/metabolism , Mice , Mice, Inbred ICR
5.
Microsc Res Tech ; 74(5): 421-9, 2011 May.
Article in English | MEDLINE | ID: mdl-20830702

ABSTRACT

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4) -induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis.


Subject(s)
Chemokine CCL4/toxicity , Connexin 43/deficiency , Liver Cirrhosis/chemically induced , Liver/pathology , Pathology, Molecular , Animals , Mice
6.
Liver Int ; 31(7): 932-9, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21092070

ABSTRACT

BACKGROUND: We investigated the reversibility of liver fibrosis induced with a CCl(4) injection and the role of stem cells in reversing the hepatic injury. Furthermore, the most effective cell fraction among bone marrow cells (BMCs) in the repair process was analysed. METHODS: C57BL/6 mice were divided into four groups after 5 weeks of injection of CCl(4) : control, sacrificed after 5 weeks, sacrificed at 10 weeks and sacrificed 5 weeks later after GFP-donor BM transplantation. Liver function tests and real-time polymerase chain reaction (PCR) of markers indicating liver fibrosis were compared between the groups. To identify the most effective BMC fraction that repairs liver injury, the mice were divided into three groups after the injection of CCl(4) for 2 days: granulocyte colony stimulating factor (G-CSF) only, mononuclear cell (MNC) transplantation and Lin-Sca-1+c-kit+haematopoietic stem cell (HSC) transplantation. Eight days after transplantation, the mice were harvested and morphometric, immunohistochemical analyses were performed to compare the expression of extracellular matrix and liver fibrosis-related factors. RESULTS: The liver fibrosis induced by CCl(4) was not spontaneously recovered but was persistent until 10 weeks, but the group injected with BMCs had less fibrosis and better liver function. Mobilization with G-CSF increased the recovery of the injured liver and the best results were seen in those mice administered the MNC fraction and Lin-Sca-1+c-kit+HSC fraction, with no difference between the two groups. CONCLUSION: BMC transplantation and stem cell mobilization with G-CSF effectively treats liver injury in mice. These are promising techniques for autologous transplantation in humans with liver fibrosis.


Subject(s)
Bone Marrow Transplantation/methods , Liver Cirrhosis, Experimental/therapy , Animals , Apoptosis , Chemokine CCL4/toxicity , DNA Primers/genetics , Granulocyte Colony-Stimulating Factor/administration & dosage , Hematopoietic Stem Cell Transplantation , Histological Techniques , Immunohistochemistry , Leukocytes, Mononuclear/transplantation , Liver Cirrhosis, Experimental/chemically induced , Liver Function Tests , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction
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