ABSTRACT
Bone marrow mesenchymal stem cells (BMSCs) transplantation has attracted attention for the treatment of liver cirrhosis and end-stage liver diseases. Therefore, in this study, we evaluated the effect of different methods of BMSCs transplantation in the treatment of liver cirrhosis in rats. Seventy-two male Sprague-Dawley rats were divided into 7 groups: 10 were used to extract BMSCs, 10 were used as normal group, and the remaining 52 rats were randomly divided into five groups for testing: control group, BMSCs group, BMSCs+granulocyte colony-stimulating factor (G-CSF) group, and BMSCs+Jisheng Shenqi decoction (JSSQ) group. After the end of the intervention course, liver tissue sections of rats were subjected to hematoxylin and eosin (H&E) and Masson staining, and pathological grades were scored. Liver function [aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB)] and hepatic fibrosis markers [hyaluronidase (HA), laminin (LN), type III procollagen (PCIII), type IV collagen (CIV)] were measured. BMSCs+JSSQ group had the best effect of reducing ALT and increasing ALB after intervention therapy (P<0.05). The reducing pathological scores and LN, PCIII, CIV of BMSCs+G-CSF group and BMSCs+JSSQ group after intervention therapy were significant, but there was no significant difference between the two groups (P>0.05). The effect of JSSQ on improving stem cell transplantation in rats with liver cirrhosis was confirmed. JSSQ combined with BMSCs could significantly improve liver function and liver pathology scores of rats with liver cirrhosis.
Subject(s)
Liver Cirrhosis, Experimental/surgery , Mesenchymal Stem Cell Transplantation/methods , Alanine Transaminase/blood , Animals , Aspartate Aminotransferases/blood , Liver Cirrhosis, Experimental/pathology , Male , Rats , Rats, Sprague-DawleyABSTRACT
OBJECTIVES: Liver transplantation is the well-known treatment for chronic liver diseases; however, postoperative complications and lack of donors continue to be limitations with this treatment. Investigating new modalities for treatment of chronic liver illness is a must. In the present study, we aimed to clarify the effects of an in vitro hepatocyte-differentiated human unrestricted somatic stem cell transplant as a new cell-based therapy in an experimental model of chronic liver failure. MATERIALS AND METHODS: Human umbilical cord blood-derived unrestricted somatic stem cells were isolated, cultured, propagated, and characterized. Cells were directed to differentiate into hepatocyte-like cells. An animal model of carbon tetrachloride cirrhotic liver failure was prepared, and the human in vitro differentiated unrestricted somatic stem cells were transplanted into the experimental model. Animals that did not receive transplant served as the pathologic control group. Animals were euthanized 12 weeks after transplant, and liver functions and histopathology were assessed. RESULTS: Compared with the pathologic control group, the transplant group showed improvements in levels of alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin. Histopathologic examination of the transplant group also showed improvements in hydropic degeneration and fibrosis. CONCLUSIONS: The use of unrestricted somatic stem cells, isolated and propagated from cord blood and then differentiated into hepatocyte-like cells, improved both fibrosis and normal function of cirrhotic livers. These cells could be considered as a line of cell-based therapy in cases of chronic liver disease.
Subject(s)
Adult Stem Cells/transplantation , Chemical and Drug Induced Liver Injury/surgery , Cord Blood Stem Cell Transplantation/methods , Fetal Blood/cytology , Hepatocytes/transplantation , Liver Cirrhosis, Experimental/surgery , Liver Regeneration , Liver/pathology , Adult Stem Cells/metabolism , Animals , Biomarkers/metabolism , Carbon Tetrachloride , Cell Differentiation , Cell Proliferation , Cells, Cultured , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Hepatocytes/metabolism , Humans , Liver/metabolism , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , Mice, Inbred BALB C , Phenotype , Time FactorsABSTRACT
Bone marrow mesenchymal stem cells (BMSCs) transplantation has attracted attention for the treatment of liver cirrhosis and end-stage liver diseases. Therefore, in this study, we evaluated the effect of different methods of BMSCs transplantation in the treatment of liver cirrhosis in rats. Seventy-two male Sprague-Dawley rats were divided into 7 groups: 10 were used to extract BMSCs, 10 were used as normal group, and the remaining 52 rats were randomly divided into five groups for testing: control group, BMSCs group, BMSCs+granulocyte colony-stimulating factor (G-CSF) group, and BMSCs+Jisheng Shenqi decoction (JSSQ) group. After the end of the intervention course, liver tissue sections of rats were subjected to hematoxylin and eosin (H&E) and Masson staining, and pathological grades were scored. Liver function [aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB)] and hepatic fibrosis markers [hyaluronidase (HA), laminin (LN), type III procollagen (PCIII), type IV collagen (CIV)] were measured. BMSCs+JSSQ group had the best effect of reducing ALT and increasing ALB after intervention therapy (P<0.05). The reducing pathological scores and LN, PCIII, CIV of BMSCs+G-CSF group and BMSCs+JSSQ group after intervention therapy were significant, but there was no significant difference between the two groups (P>0.05). The effect of JSSQ on improving stem cell transplantation in rats with liver cirrhosis was confirmed. JSSQ combined with BMSCs could significantly improve liver function and liver pathology scores of rats with liver cirrhosis.
Subject(s)
Animals , Male , Rats , Mesenchymal Stem Cell Transplantation/methods , Liver Cirrhosis, Experimental/surgery , Aspartate Aminotransferases/blood , Rats, Sprague-Dawley , Alanine Transaminase/blood , Liver Cirrhosis, Experimental/pathologyABSTRACT
AIM: Major hepatic resection of a cirrhotic liver may result in a fatal clinical course. Preoperative erythropoietin (EPO) treatment has been shown to have protective properties and to stimulate liver regeneration. This study aims to investigate the effect of preoperative EPO on survival following major hepatic resection in a cirrhotic rat model. METHODS: Cirrhotic liver was induced by intraperitoneal injection of thioacetamide (200mg/kg/mL) in 72 Lewis rats. Each 36 rats received EPO (1IU/g, every second day, 5 times preoperatively) or saline (control) and major hepatectomy (removal of the left and half of the median lobe) was performed. Biochemical and immunohistochemical parameters, cytokines and overall survival were compared following surgery. RESULTS: Rats that received preoperative EPO had decreased hepatic aspartate aminotransferase, alanine aminotransferase and interleukin (IL)-1ß expression, 48hours following surgery. They had increased hepatocyte growth factor and vascular endothelial growth factor expression at 1hour, increased IL-6 expression at 24, 48 and 120hours and increased Ki-67, 120hours following surgery. Overall, survival was significantly improved among EPO-treated rats (P=0.034). CONCLUSION: Preoperative EPO treatment has a protective effect and stimulates liver regeneration, leading to improved overall survival following major hepatectomy in a cirrhotic rat model.
Subject(s)
Erythropoietin/pharmacology , Hepatectomy/mortality , Liver Cirrhosis, Experimental/surgery , Premedication , Alanine Transaminase/blood , Animals , Aspartate Aminotransferases/blood , Disease Models, Animal , Hepatocyte Growth Factor/genetics , Hepatocyte Growth Factor/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Ki-67 Antigen/blood , Liver Regeneration , RNA, Messenger/metabolism , Rats, Inbred Lew , Survival Analysis , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
OBJECTIVES: Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. MATERIALS AND METHODS: Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. RESULTS: Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. CONCLUSIONS: Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.
Subject(s)
Cell Differentiation , Chemical and Drug Induced Liver Injury/surgery , Cord Blood Stem Cell Transplantation , End Stage Liver Disease/surgery , Hepatocytes/transplantation , Liver Cirrhosis, Experimental/surgery , Liver Transplantation/methods , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Animals , Biomarkers/metabolism , Carbon Tetrachloride , Cells, Cultured , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/pathology , Chemical and Drug Induced Liver Injury/physiopathology , End Stage Liver Disease/chemically induced , End Stage Liver Disease/pathology , End Stage Liver Disease/physiopathology , Hepatocytes/metabolism , Humans , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/pathology , Liver Cirrhosis, Experimental/physiopathology , Liver Regeneration , Male , Mesenchymal Stem Cells/metabolism , Mice, Inbred BALB C , Phenotype , Rats, Inbred Lew , Recovery of Function , Time FactorsABSTRACT
Liver transplantation is the only therapeutic treatment for patients with end-stage liver diseases. However, donor organ scarcity is the major limitation, and therefore, alternative strategies are urgently needed. The ultimate goal for successful cell-based therapies is the ability of transplanted cells to efficiently engraft and reconstitute injured liver mass. To evaluate the repopulation capacity of transplanted cells, it is essential to identify their specific characteristics, as well as to study the mechanism(s) Through which transplanted donor cells replace tissue mass in hepatic microenvironments, using well-established cell transplantation models. To date, rat fetal liver stem/progenitor cells represent the most efficient cell population to reconstitute the near-normal liver and the liver microenvironment with advanced fibrosis/cirrhosis, and therefore, can be used for developing strategies in engineering potential donor cells in the future that will be useful for clinical application in hepatic cell therapy.The present protocol describes the isolation of epithelial stem/progenitor cells derived from ED14/15 fetal livers of DPPIV+ F344 or F344-Tg(EGFP) F455/Rrrc rats, the immunohistochemical staining method to detect E-cadherin-positive epithelial cells within unfractionated cell isolates, their transplantation into different DPPIV- liver microenvironments (near-normal, retrorsine-treated, and TAA-induced fibrotic/cirrhotic liver), as well as detection methods to follow the fate of transplanted cells in the recipient liver (see Fig. 1).
Subject(s)
Cell Separation/methods , Epithelial Cells/transplantation , Fetal Stem Cells/transplantation , Hepatocytes/transplantation , Liver Cirrhosis, Experimental/surgery , Stem Cell Transplantation/methods , Animals , Cadherins/metabolism , Cell Differentiation/drug effects , Epithelial Cells/physiology , Female , Fetal Stem Cells/physiology , Hepatocytes/drug effects , Hepatocytes/physiology , Humans , Immunohistochemistry/methods , Liver/cytology , Liver/drug effects , Liver/surgery , Liver Cirrhosis/chemically induced , Liver Cirrhosis/surgery , Liver Cirrhosis, Experimental/chemically induced , Male , Pyrrolizidine Alkaloids/pharmacology , Rats , Rats, Inbred F344 , Thioacetamide/toxicityABSTRACT
Purpose. To investigate the preventive effect of resveratrol (RES) on the formation of portal vein system thrombosis (PVST) in a rat fibrosis model. Methods. A total of 64 male SD rats, weighing 200-300 g, were divided into five groups: Sham operation, Splenectomy I, Splenectomy II, RES, and low molecular weight heparin (LMWH), with the former two groups as nonfibrosis controls. Blood samples were subjected to biochemical assays. Platelet apoptosis was measured by flow cytometry. All rats were euthanized for PVST detection one week after operation. Results. No PVST occurred in nonfibrosis controls. Compared to Splenectomy II, the incidences of PVST in RES and LMWH groups were significantly decreased (both p < 0.05). Two rats in LMWH group died before euthanasia due to intra-abdominal hemorrhage. In RES group, significant decreases in platelet aggregation, platelet radical oxygen species (ROS) production, and increase in platelet nitric oxide (NO) synthesis and platelet apoptosis were observed when compared with Splenectomy II (all p < 0.001), while in LMWH group only significant decrease in platelet aggregation was observed. Conclusion. Prophylactic application of RES could safely reduce the incidence of PVST after splenectomy in cirrhotic rat. Regulation of platelet function and induction of platelet apoptosis might be the underlying mechanisms.
Subject(s)
Fibrinolytic Agents/pharmacology , Liver Cirrhosis, Experimental/drug therapy , Liver Cirrhosis, Experimental/surgery , Portal Vein , Splenectomy/adverse effects , Stilbenes/pharmacology , Venous Thrombosis/prevention & control , Animals , Anticoagulants/pharmacology , Apoptosis/drug effects , Blood Platelets/drug effects , Blood Platelets/metabolism , Blood Platelets/pathology , Enoxaparin/pharmacology , Liver Cirrhosis, Experimental/complications , Male , Nitric Oxide/metabolism , Platelet Aggregation/drug effects , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Resveratrol , Venous Thrombosis/etiologyABSTRACT
AIM: To evaluate the therapeutic effects of bone marrow-derived mesenchymal stem cells (BMSCs) with human urokinase-type plasminogen activator (uPA) on liver fibrosis, and to investigate the mechanism of gene therapy. METHODS: BMSCs transfected with adenovirus-mediated human urokinase plasminogen activator (Ad-uPA) were transplanted into rats with CCl4-induced liver fibrosis. All rats were sacrificed after 8 wk, and their serum and liver tissue were collected for biochemical, histopathologic, and molecular analyzes. The degree of liver fibrosis was assessed by hematoxylin and eosin or Masson's staining. Western blot and quantitative reverse transcription-polymerase chain reaction were used to determine protein and mRNA expression levels. RESULTS: Serum levels of alanine aminotransferase, aminotransferase, total bilirubin, hyaluronic acid, laminin, and procollagen type III were markedly decreased, whereas the levels of serum albumin were increased by uPA gene modified BMSCs treatment. Histopathology revealed that chronic CCl4-treatment resulted in significant fibrosis while uPA gene modified BMSCs treatment significantly reversed fibrosis. By quantitatively analysing the fibrosis area of liver tissue using Masson staining in different groups of animals, we found that model animals with CCl4-induced liver fibrosis had the largest fibrotic area (16.69% ± 1.30%), while fibrotic area was significantly decreased by BMSCs treatment (12.38% ± 2.27%) and was further reduced by uPA-BMSCs treatment (8.31% ± 1.21%). Both protein and mRNA expression of ß-catenin, Wnt4 and Wnt5a was down-regulated in liver tissues following uPA gene modified BMSCs treatment when compared with the model animals. CONCLUSION: Transplantation of uPA gene modified BMSCs suppressed liver fibrosis and ameliorated liver function and may be a new approach to treating liver fibrosis. Furthermore, treatment with uPA gene modified BMSCs also resulted in a decrease in expression of molecules of the Wnt signaling pathway.
Subject(s)
Chemical and Drug Induced Liver Injury/surgery , Genetic Therapy/methods , Liver Cirrhosis, Experimental/surgery , Liver/enzymology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/enzymology , Urokinase-Type Plasminogen Activator/biosynthesis , Wnt Signaling Pathway , Adenoviridae/genetics , Animals , Carbon Tetrachloride , Cells, Cultured , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/pathology , Enzyme Induction , Genetic Vectors , Humans , Liver/pathology , Liver Cirrhosis, Experimental/enzymology , Liver Cirrhosis, Experimental/genetics , Liver Cirrhosis, Experimental/pathology , Male , Rats, Sprague-Dawley , Transfection , Urokinase-Type Plasminogen Activator/genetics , Wnt Signaling Pathway/genetics , Wnt-5a Protein/genetics , Wnt-5a Protein/metabolism , Wnt4 Protein/genetics , Wnt4 Protein/metabolism , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
BACKGROUND & AIMS: Clinical studies suggest that splenectomy improves liver function in cirrhotic patients, but the influence of splenectomy on stem cell transplantation is poorly understood. This study investigated the effect of splenectomy on stem cell infusion and elucidated its mechanism. METHODS: Rat adipose tissue-derived mesenchymal stem cells were infused into cirrhosis rats with or without splenectomy, followed by the assessment of the in vivo distribution of stem cells and pathological changes. Stromal cell-derived factor-1 and hepatocyte growth factor expression were also investigated in splenectomized cirrhosis patients and rats. RESULTS: Splenectomy, prior to cell infusion, improved liver function and suppressed fibrosis progression more efficiently than cell infusion alone in the experimental cirrhosis model. Stromal cell-derived factor-1 and hepatocyte growth factor levels after splenectomy were increased in patients and rats. These upregulated cytokines significantly facilitated stem cell motility, migration and proliferation in vitro. C-X-C chemokine receptor type 4 neutralization weakened the promotion of cell migration by these cytokines. The infused cells integrated into liver fibrosis septa and participated in regeneration more efficiently in splenectomized rats. Direct coculture with stem cells led to inhibition of hepatic stellate cell proliferation. In addition, hepatocyte growth factor induced hepatic stellate cell apoptosis via the c-jun N-terminal kinase-p53 pathway. CONCLUSIONS: Splenectomy prior to cell infusion enhanced the therapeutic effect of stem cells on cirrhosis, which involved upregulation of stromal cell-derived factor-1 and hepatocyte growth factor after splenectomy.
Subject(s)
Chemokine CXCL12/metabolism , Hepatocyte Growth Factor/metabolism , Liver Cirrhosis, Experimental/therapy , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Splenectomy , Adipose Tissue/pathology , Animals , Cell Proliferation , Cells, Cultured , Hepatocytes/pathology , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Liver/pathology , Liver Cirrhosis, Experimental/surgery , Liver Regeneration , Male , Rats , Rats, Inbred F344ABSTRACT
PURPOSE: To present a review about a comparative study of bile duct ligation versus carbon tetrachloride Injection for inducing experimental liver cirrhosis. METHODS: This research was made through Medline/PubMed and SciELO web sites looking for papers on the content "induction of liver cirrhosis in rats". We have found 107 articles but only 30 were selected from 2004 to 2011. RESULTS: The most common methods used for inducing liver cirrhosis in the rat were administration of carbon tetrachloride (CCl4) and bile duct ligation (BDL). CCl4 has induced cirrhosis from 36 hours to 18 weeks after injection and BDL from seven days to four weeks after surgery. CONCLUSION: For a safer inducing cirrhosis method BDL is better than CCl4 because of the absence of toxicity for researches and shorter time for achieving it.
Subject(s)
Bile Ducts/surgery , Carbon Tetrachloride/toxicity , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/surgery , Animals , Ligation , Rats , Time FactorsABSTRACT
PURPOSE: To present a review about a comparative study of bile duct ligation versus carbon tetrachloride Injection for inducing experimental liver cirrhosis. METHODS: This research was made through Medline/PubMed and SciELO web sites looking for papers on the content "induction of liver cirrhosis in rats". We have found 107 articles but only 30 were selected from 2004 to 2011. RESULTS: The most common methods used for inducing liver cirrhosis in the rat were administration of carbon tetrachloride (CCl4) and bile duct ligation (BDL). CCl4 has induced cirrhosis from 36 hours to 18 weeks after injection and BDL from seven days to four weeks after surgery. CONCLUSION: For a safer inducing cirrhosis method BDL is better than CCl4 because of the absence of toxicity for researches and shorter time for achieving it.
OBJETIVO: Apresentar revisão sobre estudo comparativo da indução de cirrose hepática (CH) experimental com a injeção de tetra-cloreto de carbono (CCl4) comparado à ligadura do ducto biliar (BDL). MÉTODOS: A pesquisa foi realizada nas bases de dados do Medline/PubMed e SciELO procurando trabalhos com as palavras indução de CH e ratos. Foram encontrados 107 artigos, mas somente 30 foram selecionados no período de 2004 à 2011. RESULTADOS: Os procedimentos mais comum para indução de CH em ratos foram a injeção de CCl4 e a BDL. O CCl4 induzia CH no período de 36 horas após a injeção e a DBL de sete dias à quatro semanas após a cirurgia. CONCLUSÃO: A BDL é o método mais seguro para indução de CH quando comparado a injeção de CCl4 pela ausência de toxicidade para os pesquisadores e o menor tempo para se obter a lesão hepática.
Subject(s)
Animals , Rats , Bile Ducts/surgery , Carbon Tetrachloride/toxicity , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/surgery , Ligation , Time FactorsABSTRACT
OBJECTIVE: To observe the inhibitory effect of angiotensin-(1-7) on liver fibrosis induced by bile duct ligation in rats. METHODS: Eighteen Wistar rats were randomized into 3 groups and subject to sham operation, bile duct ligation (BDL), or BDL with angiotensin-(1-7) treatment. An osmotic minipump was implanted intraperitoneally for administration of saline in the sham-operated and BDL groups and angiotensin-(1-7) (25 µg·kg(-1)·h(-1)) in angiotensin-(1-7) treatment group. After a 4-week treatments, the fibrosis score, Masson staining, and hydroxyproline assay were used to evaluate the level of liver fibrosis in the rats, and immunohistochemistry was used to detect expression of α-smooth muscle actin (α-SMA) in the liver tissue. RESULTS: Compared with BDL group, a 4-week treatment with angiotensin-(1-7) following BDL significantly reduced the fibrosis score (2.33±0.52 vs 5.17±0.75), hydroxyproline content (0.36±0.03 vs 0.52±0.04) and α-SMA expression (54.11±17.55 vs 191.84±31.72) in the liver tissue of the rats (P<0.01). CONCLUSION: Prolonged infusion of angiotensin-(1-7) inhibit the formation of hepatic fibrosis in rats following bile duct ligation.
Subject(s)
Angiotensin I/pharmacology , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/prevention & control , Peptide Fragments/pharmacology , Angiotensin I/administration & dosage , Animals , Bile Ducts/surgery , Infusions, Parenteral , Ligation , Liver Cirrhosis, Experimental/surgery , Male , Peptide Fragments/administration & dosage , Rats , Rats, WistarABSTRACT
We have previously reported the efficacy and safety of autologous bone marrow cell infusion (ABMi) therapy for liver cirrhosis patients without hepatocellular carcinoma in a multicenter clinical trial. However, since liver cirrhosis is highly oncogenic, evaluation of the effects of ABMi on the mechanisms of hepatocarcinogenesis is of great importance. Therefore, frequent ABMi was performed in hepatocarcinogenic mice, and its effects on hepatocarcinogenesis were analyzed. The N-nitrosodiethylamine (DEN)/green fluorescent protein (GFP)-carbon tetrachloride (CCl(4) ) model was developed by administering DEN once, followed by repeated administration of CCl(4) intraperitoneally as for the control group. In the administration (ABMi) group, GFP-positive bone marrow cells were infused through a tail vein. The kinetics of hepatocarcinogenesis were evaluated histologically 4.5 months after DEN treatment. At 4.5 months, there was significantly lower incidence of foci and tumors in the ABMi group, and they were smaller in number, while their size was almost equal. No GFP-positive tumors were found in ABMi livers. Moreover, ABMi livers showed significantly reduced liver fibrosis, consistent with significantly lower 8-hydroxy-2'-deoxyguanosine levels, higher superoxide dismutase activity, and increased nuclear translocation of nuclear factor-erythroid 2 p45-related factor 2. These results demonstrate that frequent ABMi might contribute to suppressed tumor initiation during stages of hepatocarcinogenesis, consistent with improvements in liver fibrosis and stabilization of redox homeostasis.
Subject(s)
Bone Marrow Transplantation , Carcinoma, Hepatocellular/prevention & control , Cell Transformation, Neoplastic/pathology , Liver Cirrhosis, Experimental/surgery , Liver Neoplasms, Experimental/prevention & control , Liver/pathology , 8-Hydroxy-2'-Deoxyguanosine , Animals , Carbon Tetrachloride , Carcinoma, Hepatocellular/chemically induced , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Transformation, Neoplastic/chemically induced , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Diethylnitrosamine , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Homeostasis , Liver/metabolism , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , NF-E2-Related Factor 2/metabolism , Oxidation-Reduction , Oxidative Stress , Superoxide Dismutase/metabolism , Time Factors , Transplantation, AutologousABSTRACT
AIM: To investigate the efficacy and mechanism of action of allogeneic embryonic hepatocyte transplantation for the treatment of hepatic cirrhosis. METHODS: Rat embryonic hepatocytes were characterized by examining cell markers. Wistar rats with CCl(4)-induced cirrhosis were randomly divided into two groups: a model group receiving continuous CCl(4), and a cell transplantation group receiving continuous CCl(4) and transplanted with embryonic fluorescent-labeled hepatocytes. In addition, a normal control group was composed of healthy rats. All rats were sacrificed after 2 wk following the initiation of the cell transplant. Ultrasound, pathological analyses and serum biochemical tests were used to evaluate the efficacy of embryonic hepatocyte transplantation. To analyze the recovery status of cirrhotic hepatocytes and the signaling pathways influenced by embryonic hepatocyte transplantation, real-time polymerase chain reaction was performed to examine the mRNA expression of stellate activation-associated protein (STAP), c-myb, α smooth muscle actin (α-SMA) and endothelin-1 (ET-1). Western blotting and immunohistochemistry were employed to detect α-SMA and ET-1 protein expression in hepatic tissues. RESULTS: Gross morphological, ultrasound and histopathological examinations, serum biochemical tests and radioimmunoassays demonstrated that hepatic cirrhosis was successfully established in the Wistar rats. Stem cell factor receptor (c-kit), hepatocyte growth factor receptor (c-Met), Nestin, α fetal protein, albumin and cytokeratin19 markers were observed in the rat embryonic hepatocytes. Following embryonic hepatocyte transplantation, there was a significant reversal in the gross appearance, ultrasound findings, histopathological properties, and serum biochemical parameters of the rat liver. In addition, after the activation of hepatic stellate cells and STAP signaling, α-SMA, c-myb and ET-1 mRNA levels became significantly lower than in the untreated cirrhotic group (P < 0.05). These levels, however, were not statistically different from those of the normal healthy group. Immunohistochemical staining and Western blot analyses revealed that α-SMA and ET-1 protein expression levels in the transplantation group were significantly lower than in the untreated cirrhotic group, but being not statistically different from the normal group. CONCLUSION: Transplantation of embryonic hepatocytes in rats has therapeutic effects on cirrhosis. The described treatment may significantly reduce the expression of STAP and ET-1.
Subject(s)
Embryo, Mammalian/cytology , Hepatocytes/transplantation , Liver Cirrhosis, Experimental/surgery , Animals , Biomarkers/metabolism , Carbon Tetrachloride/toxicity , Cytoglobin , Endothelin-1/metabolism , Female , Hepatocytes/metabolism , Liver/cytology , Liver/metabolism , Liver/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , Male , Peroxidases/metabolism , Random Allocation , Rats , Rats, Wistar , Signal Transduction/physiologyABSTRACT
UNLABELLED: In advanced cirrhosis, impaired function is caused by intrinsic damage to the native liver cells and from the abnormal microenvironment in which the cells reside. The extent to which each plays a role in liver failure and regeneration is unknown. To examine this issue, hepatocytes from cirrhotic and age-matched control rats were isolated, characterized, and transplanted into the livers of noncirrhotic hosts whose livers permit extensive repopulation with donor cells. Primary hepatocytes derived from livers with advanced cirrhosis and compensated function maintained metabolic activity and the ability to secrete liver-specific proteins, whereas hepatocytes derived from cirrhotic livers with decompensated function failed to maintain metabolic or secretory activity. Telomere studies and transcriptomic analysis of hepatocytes recovered from progressively worsening cirrhotic livers suggest that hepatocytes from irreversibly failing livers show signs of replicative senescence and express genes that simultaneously drive both proliferation and apoptosis, with a later effect on metabolism, all under the control of a central cluster of regulatory genes, including nuclear factor κB and hepatocyte nuclear factor 4α. Cells from cirrhotic and control livers engrafted equally well, but those from animals with cirrhosis and failing livers showed little initial evidence of proliferative capacity or function. Both, however, recovered more than 2 months after transplantation, indicating that either mature hepatocytes or a subpopulation of adult stem cells are capable of full recovery in severe cirrhosis. CONCLUSION: Transplantation studies indicate that the state of the host microenvironment is critical to the regenerative potential of hepatocytes, and that a change in the extracellular matrix can lead to regeneration and restoration of function by cells derived from livers with end-stage organ failure.
Subject(s)
Cellular Microenvironment/physiology , Hepatocytes/physiology , Hepatocytes/transplantation , Liver Cirrhosis, Experimental/surgery , Liver Regeneration/physiology , Animals , Cell Proliferation , Cell Transplantation/methods , Cells, Cultured/physiology , Disease Models, Animal , Extracellular Matrix/metabolism , Extracellular Matrix/physiology , Liver Cirrhosis, Experimental/pathology , Male , Oligonucleotide Array Sequence Analysis , RNA, Messenger/analysis , Random Allocation , Rats , Rats, Inbred Lew , Recovery of Function , Reference Values , Severity of Illness Index , TelomereABSTRACT
BACKGROUND: The present study investigated the alteration of intestinal microbiota, innate immunity-related genes, and bacterial translocation in rats with cirrhosis and liver transplantation. METHODS: Specific pathogen-free Sprague-Dawley rats were randomized into 4 groups: (1) normal controls (N); (2) liver cirrhosis (LC); (3) normal control groups with liver transplantation (LTN); and (4) liver cirrhosis with liver transplantation (LTC). We examined plasma endotoxin, bacterial tacslocation, denaturing gradient gel electrophoresis (DGGE) profile of intestinal mucosa-associated bacteria, abundance of key bacterial populations, and expression of innate immunity-related gene. RESULTS: The LTC and LC group, showed higher endotoxin levels (1.08±0.73 EU/mL and 0.74±0.70 EU/mL, respectively) than the N group (0.27±0.13 EU/mL; P<.05). the incidence of bacterial translocation (BT) to liver and mesenteric lymph nodes (MLN), and the number of total bacteria were increased significantly in the LTC and LC groups compared with the N group (P<.05). The counts of Lactobacilli and Bacteroides were lower, whereas Enterobacteria were higher in the LC than the N group (P<.05). Mucins (MUC2, MUC3) and Toll-like receptors (TLR2, TLR4) messenger RNA (mRNA) expression were significantly higher in the LC and LTC groups than the N group (P<.05). The marked difference between the groups in the overall structure of the bacterial community was also generated by DGGE profiles. CONCLUSION: Liver cirrhosis disturbs intestinal microbiota and innate immunity-related genes, which contributes to endotoxemia and bacterial translocation. These had not completely recovered in cirrhotic rats until 1 month after orthotopic liver transplantation.
Subject(s)
Bacterial Translocation , Ileum/microbiology , Immunity, Innate/genetics , Intestines/microbiology , Liver Cirrhosis, Experimental/surgery , Liver Transplantation , Animals , Carbon Tetrachloride , Denaturing Gradient Gel Electrophoresis , Endotoxins/blood , Gene Expression Regulation , Ileum/immunology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/genetics , Liver Cirrhosis, Experimental/immunology , Liver Cirrhosis, Experimental/microbiology , Male , Mucin-2/genetics , Mucin-3/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Time Factors , Toll-Like Receptor 2/genetics , Toll-Like Receptor 4/geneticsABSTRACT
Oxidative stress in liver cells may contribute to the etiology of hepatic diseases, as in liver cirrhosis. AP-Endonuclease1 (APE1/Ref-1) is essential for cell protection toward oxidative stress by acting as a transcriptional regulator of pro-survival genes and as a redox sensitive protein. The aim of this study was to critically analyze the various parameters governing the success of human umbilical cord blood mononuclear stem cell-based (MNCs) therapy without the use of an immunosuppressant and to investigate for the first time the expression of APE1 during thioacetamide (TAA)-induced cirrhosis and MNCs therapy in a rat model. Umbilical cord blood samples from full-term deliveries were collected. Lethal fulminant hepatic cirrhosis in rats was induced by intraperitoneal injection of thio-acetamide. MNCs were then intrahepatically transplanted. We measured APE1 expression at mRNA and protein levels, mRNA expression of TGF-ß, α-SMA, STAP, CTGF, MMP-9 and TIMP-1 in a follow up study. Histopathological and immunohistochemical analyses were performed 10 weeks after intrahepatic injection of the cells. Transdifferentiated cells could be efficiently stained with antihuman hepatocytes. Interestingly, human hepatocyte-specific markers, human albumin, cytokeratin-18 and cytokeratin-19 mRNAs were detected in rat liver after 10 days of MNCs infusion. MNC transplanted by intrahepatic route, could engraft recipient liver, differentiated into functional hepatocytes, and rescued liver failure. Moreover up regulation of APE1 expression confirmed by marked immunohistochemical staining may be involved in MNCs-induced hepatocytes regeneration suggesting that maintaining high level of APE1 has protective effect as pro-survival signal.
Subject(s)
Cord Blood Stem Cell Transplantation , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Liver Cirrhosis, Experimental/surgery , Liver Regeneration , Liver/surgery , Actins/genetics , Animals , Connective Tissue Growth Factor/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , Gene Expression Regulation, Enzymologic , Heat-Shock Proteins/genetics , Humans , Immunohistochemistry , Keratin-18/genetics , Keratin-19/genetics , Lipid Peroxidation , Liver/enzymology , Liver/pathology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/enzymology , Liver Cirrhosis, Experimental/genetics , Liver Cirrhosis, Experimental/pathology , Male , Matrix Metalloproteinase 9/genetics , Oxidative Stress , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Sequestosome-1 Protein , Serum Albumin/genetics , Thioacetamide , Time Factors , Tissue Inhibitor of Metalloproteinase-1/genetics , Transforming Growth Factor beta/geneticsABSTRACT
The possibility to recover the antioxidant system in rats with experimental liver cirrhosis (LC) after allo- and xenotransplantation of cryopreserved fetal liver cells (FLC) was investigated. It was shown that the content of lipid peroxidation products in the blood serum of animals with LC four weeks after FLC transplantation decreased significantly as compared to control group. Such changes were accompanied by a significant increase of catalase (CAT), glutathione reductase (GR), Se-dependent glutathione peroxidase (GP) activity in the liver and total anti-oxidative activity (AOA) of blood. Obtained results demonstrate that the main direction of FLC effects in animals with LC agree with that we observed previously in other experimental models (partial hepatectomy, chronic alcohol poisoning and hypercholesterolemia). In conclusion, cell therapy may be considered as the universal method for correction of disorders in regulation of free-radical processes in various experimental pathologies.
Subject(s)
Antioxidants/metabolism , Cell Transplantation , Cryopreservation , Liver Cirrhosis, Experimental/surgery , Liver/cytology , Animals , Fetus , Humans , Lipid Peroxides/blood , Lipid Peroxides/metabolism , Liver/embryology , Liver/enzymology , Liver/metabolism , Liver Cirrhosis, Experimental/blood , Liver Cirrhosis, Experimental/enzymology , Male , Rats , Transplantation, Heterologous , Transplantation, Homologous , Treatment OutcomeABSTRACT
BACKGROUND: The regeneration capacity of cirrhotic livers might be affected by angiotensin-1 (AT1) receptors located on hepatic stellate cells (HSC). The effect of AT1 receptor blockade on microcirculation, fibrosis and liver regeneration was investigated. MATERIALS AND METHODS: In 112 Lewis rats, cirrhosis was induced by repetitive intraperitoneal injections of CCl(4) . Six hours, 3, 7 and 14 days after partial hepatectomy or sham operation, rats were sacrificed for analysis. Animals were treated with either vehicle or 5 mg/kg body weight losartan pre-operatively and once daily after surgery by gavage. Microcirculation and portal vein flow were investigated at 6 h. The degree of cirrhosis was assessed by Azan Heidenhein staining, activation of HSC by desmin staining, apoptosis by ssDNA detection and liver regeneration by Ki-67 staining. Changes in expression of various genes important for liver regeneration and fibrosis were analysed at 6 h and 3 days. Haemodynamic parameters and liver enzymes were monitored. RESULTS: Losartan treatment increased sinusoidal diameter, sinusoidal blood flow and portal vein flow after partial hepatectomy (P<0.05), but not after sham operation. AT1 receptor blockade resulted in increased apoptosis early after resection. HSC activation was reduced and after 7 days, a significantly lower degree of cirrhosis in resected animals was observed. Losartan increased the proliferation of hepatocytes at late time-points and of non-parenchymal cells early after partial hepatectomy (P<0.05). Tumour necrosis factor (TNF)-α was significantly upregulated at 6 h and stem cell growth factor (SCF) was downregulated at 3 days (P<0.05). CONCLUSION: Losartan increased hepatic blood flow, reduced HSC activation and liver fibrosis, but interfered with hepatocyte proliferation after partial hepatectomy in cirrhotic livers.