AIM:To investigate the role of hepatocyte growth factor(HGF) in cholangiocarcinoma(CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS:Effects of HGF on cell invasion and motility wer...AIM:To investigate the role of hepatocyte growth factor(HGF) in cholangiocarcinoma(CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS:Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines,HuCCA-1 and KKU-M213,using Transwell in vitro assay.Levels of proteins of interest and their phosphorylated forms were determined by Western blotting.Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography. RESULTS:Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line.HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization,but did not affect the levels of secreted matrix metalloproteinase(MMP) -2,MMP-9 andurokinase plasminogen activator,key matrix degrading enzymes involved in cell invasion.Concomitantly,HGF stimulated Akt and extracellular signal-regulated kinase(ERK) 1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines.Inhibition of the phosphoinositide 3-kinase(PI3K) /Akt pathway by the PI3K inhibitor,LY294002,markedly suppressed HGFstimulated invasion of both CCA cell lines,and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells. CONCLUSION:These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type.展开更多
BACKGROUND: Because of an increasing discrepancy be-tween the number of potential liver graft recipients and the number of organs available, scientists are trying to create artiifcial liver to mimic normal liver funct...BACKGROUND: Because of an increasing discrepancy be-tween the number of potential liver graft recipients and the number of organs available, scientists are trying to create artiifcial liver to mimic normal liver function and therefore, to support the patient’s liver when in dysfunction. 3D printing technique meets this purpose. The present study was to test the feasibility of 3D hydrogel scaffolds for liver engineering. METHODS: We fabricated 3D hydrogel scaffolds with a bioprinter. The biocompatibility of 3D hydrogel scaffolds was tested. Sixty nude mice were randomly divided into four groups, with 15 mice in each group: control, hydrogel, hydro-gel with L02 (cell line HL-7702), and hydrogel with hepatocyte growth factor (HGF). Cells were cultured and deposited in scaffolds which were subsequently engrafted into livers after partial hepatectomy and radiation-induced liver damage (RILD). The engrafted tissues were examined after two weeks. The levels of alanine aminotransferase (ALT), aspartate amino-transferase (AST), albumin, total bilirubin, CYP1A2, CYP2C9, glutathione S-transferase (a-GST), and UDP-glucuronosyl transferase (UGT-2) were compared among the groups. He-matoxylin-eosin (HE) staining and immunohistochemistry of cKit and cytokeratin 18 (CK18) of engrafted tissues were evalu-ated. The survival time of the mice was also compared among the four groups. RESULTS: 3D hydrogel scaffolds did not impact the viability of cells. The levels of ALT, AST, albumin, total bilirubin, CY-P1A2, CYP2C9, a-GST and UGT-2 were signiifcantly improved in mice engrafted with 3D scaffold loaded with L02 compared with those in control and scaffold only (P<0.05). HE staining showed clear liver tissue and immunohistochemistry of cKit and CK18 were positive in the engrafted tissue. Mice treated with 3D scaffold+L02 cells had longer survival time compared with those in control and scaffold only (P<0.05). CONCLUSION: 3D scaffold has the potential of recreating liver tissue and partial liver functions and can be used in the reconstruction of liver tissues.展开更多
Background:Nonalcoholic fatty liver disease(NAFLD)is a global health concern with the acid sphingomyelinase(ASM)/ceramide(CE)pathway and the NOD-like receptor family,pyrin domain-containing protein 3(NLRP3)inflammasom...Background:Nonalcoholic fatty liver disease(NAFLD)is a global health concern with the acid sphingomyelinase(ASM)/ceramide(CE)pathway and the NOD-like receptor family,pyrin domain-containing protein 3(NLRP3)inflammasome identified as pivotal players in lipid disorders and inflammation.This study explores the interaction mechanism between the ASM/CE pathway and NLRP3 in NAFLD cell models,aiming to understand the impact of amitriptyline(Ami),an ASM inhibitor,on lipid deposition and hepatocyte injury by regulating the ASM/CE-NLRP3 pathway.Methods:HepG2 and HL-7702 cells were exposed to free fatty acids(FFAs)to establish the NAFLD model.The cells were divided into 5 groups:control group,model group,Ami group,tumor necrosis factoralpha(TNF-α)group,and Ami+TNF-αgroup.Intracellular lipid droplets were visualized using Oil Red O staining,and Western blot analysis quantified ASM,NLRP3,and caspase 1 protein expression.Enzyme linked immunosorbent assay(ELISA)was measured CE and ASM levels,while qRT-PCR assessed mRNA expression.The apoptotic rate was evaluated by flow cytometry(FCM).Results:Following FFAs incubation,significant increases in ASM and CE levels were observed in HepG2 and HL-7702 cells,accompanied by elevated expression of NLRP3,and caspase 1,and IL-1β.TNF-αtreatment further amplified these indicators.Ami demonstrated a reduction in lipid deposition,suppressed ASM/CE pathway activation,downregulated NLRP3 and caspase 1 expression,and improved apoptosis.Additionally,MCC950,a selective inhibitor of the NLRP3,mitigated NLRP3,caspase 1,and IL-1βexpression,alleviating lipid deposition and apoptosis in the NAFLD cell model.Conclusion:The ASM/CE-NLRP3 pathway in NAFLD cells promotes hepatocyte steatosis,inflammation,and cell damage.Ami emerges as a promising therapeutic agent by inhibiting the ASM/CE-NLRP3 pathway,underscoring its potential as a key target for NAFLD treatment.展开更多
Excessive iron is toxic to cells and organelles, where it can generate harmful reactive oxygen species (ROS) resulting in oxidative tissue damage. Liver is the major organ for iron storage and redox active iron in thi...Excessive iron is toxic to cells and organelles, where it can generate harmful reactive oxygen species (ROS) resulting in oxidative tissue damage. Liver is the major organ for iron storage and redox active iron in this tissue can cause fibrosis and cirrhosis in β-thalassemia patients. Desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) are clinically approved iron chelators used for the treatment of patients with iron overload, but none of these chelators are completely free of side effects. In this study we report the properties of a new iron chelator 1-(N-acetyl-6-aminohexyl)-3-hydroxypyridin-4-one (CM1). The labile iron pool (LIP) content was measured by using a calcein fluorescence technique and the lipidperoxidation products were quantified using the thiobarbituric acid reactive substances (TBARS) method. The cytotoxicity of CM1 was also examined with the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. CM1 was demonstrated to reduce iron-induced redox damage and to decrease the levels of the intracellular iron pool in hepatocytes. CM1 is a potentially useful iron-chelating agent which has potential to ameliorate liver iron overload and ROS-induced lipid peroxidation. CM1 is currently under investigation for oral efficacy.展开更多
In order to explore the role of forkhead box protein O1(FoxO1)in the lipid metabolism and cell proliferation,goose primary hepatocytes were isolated and incubated with insulin or PI3K-Akt-mTOR pathway dual inhibitor N...In order to explore the role of forkhead box protein O1(FoxO1)in the lipid metabolism and cell proliferation,goose primary hepatocytes were isolated and incubated with insulin or PI3K-Akt-mTOR pathway dual inhibitor NVPBEZ235,and then transfected with FoxO1 interference plasmid.The related parameters of lipid metabolism and cell proliferation were measured.The results firstly showed that FoxO1 interference increased the intracellular TG and lipids concentration(P<0.05);and increased the proliferative index(PI),cell DNA synthesis,protein expression of Cyclin D1 in goose primary hepatocytes(P<0.05).Secondly,the co-treatment of insulin and FoxO1 interference increased the mRNA level and protein content of Cyclin D1(P<0.05);however,there was no significant difference between the insulin treatment and the co-treatment of insulin and miR-FoxO1 interference in the intracellular TG and lipids concentration and PI(P>0.05).Lastly,the decrease of intracellular TG and lipids concentration and PI induced by NVP-BEZ235 was up-regulated by FoxO1 interference significantly(P<0.05).In summary,FoxO1 could regulate the lipids metabolism and cell proliferation mediated by PI3K-Akt-mTOR signaling pathway in goose primary hepatocytes.Further investigations are required to highlight the potential role of FoxO1 in the lipid metabolism and cell proliferation mediated by insulin in goose primary hepatocyte.展开更多
基金Supported by Mahidol University,Thailand and Thailand Research Fund(Suthiphongchai T)Strategic Consortia for Capacity Building of University Faculties and Staff Scholarship,Commission on Higher Education,Ministry of Education,Thailand(Menakongka A)
文摘AIM:To investigate the role of hepatocyte growth factor(HGF) in cholangiocarcinoma(CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS:Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines,HuCCA-1 and KKU-M213,using Transwell in vitro assay.Levels of proteins of interest and their phosphorylated forms were determined by Western blotting.Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography. RESULTS:Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line.HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization,but did not affect the levels of secreted matrix metalloproteinase(MMP) -2,MMP-9 andurokinase plasminogen activator,key matrix degrading enzymes involved in cell invasion.Concomitantly,HGF stimulated Akt and extracellular signal-regulated kinase(ERK) 1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines.Inhibition of the phosphoinositide 3-kinase(PI3K) /Akt pathway by the PI3K inhibitor,LY294002,markedly suppressed HGFstimulated invasion of both CCA cell lines,and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells. CONCLUSION:These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type.
基金supported by grants from the National Natural Science Foundation of Major Research and Development Plan of China(91542205)151 Talents Project of Zhejiang Province(12-1-058)
文摘BACKGROUND: Because of an increasing discrepancy be-tween the number of potential liver graft recipients and the number of organs available, scientists are trying to create artiifcial liver to mimic normal liver function and therefore, to support the patient’s liver when in dysfunction. 3D printing technique meets this purpose. The present study was to test the feasibility of 3D hydrogel scaffolds for liver engineering. METHODS: We fabricated 3D hydrogel scaffolds with a bioprinter. The biocompatibility of 3D hydrogel scaffolds was tested. Sixty nude mice were randomly divided into four groups, with 15 mice in each group: control, hydrogel, hydro-gel with L02 (cell line HL-7702), and hydrogel with hepatocyte growth factor (HGF). Cells were cultured and deposited in scaffolds which were subsequently engrafted into livers after partial hepatectomy and radiation-induced liver damage (RILD). The engrafted tissues were examined after two weeks. The levels of alanine aminotransferase (ALT), aspartate amino-transferase (AST), albumin, total bilirubin, CYP1A2, CYP2C9, glutathione S-transferase (a-GST), and UDP-glucuronosyl transferase (UGT-2) were compared among the groups. He-matoxylin-eosin (HE) staining and immunohistochemistry of cKit and cytokeratin 18 (CK18) of engrafted tissues were evalu-ated. The survival time of the mice was also compared among the four groups. RESULTS: 3D hydrogel scaffolds did not impact the viability of cells. The levels of ALT, AST, albumin, total bilirubin, CY-P1A2, CYP2C9, a-GST and UGT-2 were signiifcantly improved in mice engrafted with 3D scaffold loaded with L02 compared with those in control and scaffold only (P<0.05). HE staining showed clear liver tissue and immunohistochemistry of cKit and CK18 were positive in the engrafted tissue. Mice treated with 3D scaffold+L02 cells had longer survival time compared with those in control and scaffold only (P<0.05). CONCLUSION: 3D scaffold has the potential of recreating liver tissue and partial liver functions and can be used in the reconstruction of liver tissues.
基金supported by the Initial Scientific Research Fund of the Talents Introduced in Nanjing Lishui People’s Hospital(Project 2021YJ02).
文摘Background:Nonalcoholic fatty liver disease(NAFLD)is a global health concern with the acid sphingomyelinase(ASM)/ceramide(CE)pathway and the NOD-like receptor family,pyrin domain-containing protein 3(NLRP3)inflammasome identified as pivotal players in lipid disorders and inflammation.This study explores the interaction mechanism between the ASM/CE pathway and NLRP3 in NAFLD cell models,aiming to understand the impact of amitriptyline(Ami),an ASM inhibitor,on lipid deposition and hepatocyte injury by regulating the ASM/CE-NLRP3 pathway.Methods:HepG2 and HL-7702 cells were exposed to free fatty acids(FFAs)to establish the NAFLD model.The cells were divided into 5 groups:control group,model group,Ami group,tumor necrosis factoralpha(TNF-α)group,and Ami+TNF-αgroup.Intracellular lipid droplets were visualized using Oil Red O staining,and Western blot analysis quantified ASM,NLRP3,and caspase 1 protein expression.Enzyme linked immunosorbent assay(ELISA)was measured CE and ASM levels,while qRT-PCR assessed mRNA expression.The apoptotic rate was evaluated by flow cytometry(FCM).Results:Following FFAs incubation,significant increases in ASM and CE levels were observed in HepG2 and HL-7702 cells,accompanied by elevated expression of NLRP3,and caspase 1,and IL-1β.TNF-αtreatment further amplified these indicators.Ami demonstrated a reduction in lipid deposition,suppressed ASM/CE pathway activation,downregulated NLRP3 and caspase 1 expression,and improved apoptosis.Additionally,MCC950,a selective inhibitor of the NLRP3,mitigated NLRP3,caspase 1,and IL-1βexpression,alleviating lipid deposition and apoptosis in the NAFLD cell model.Conclusion:The ASM/CE-NLRP3 pathway in NAFLD cells promotes hepatocyte steatosis,inflammation,and cell damage.Ami emerges as a promising therapeutic agent by inhibiting the ASM/CE-NLRP3 pathway,underscoring its potential as a key target for NAFLD treatment.
文摘Excessive iron is toxic to cells and organelles, where it can generate harmful reactive oxygen species (ROS) resulting in oxidative tissue damage. Liver is the major organ for iron storage and redox active iron in this tissue can cause fibrosis and cirrhosis in β-thalassemia patients. Desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) are clinically approved iron chelators used for the treatment of patients with iron overload, but none of these chelators are completely free of side effects. In this study we report the properties of a new iron chelator 1-(N-acetyl-6-aminohexyl)-3-hydroxypyridin-4-one (CM1). The labile iron pool (LIP) content was measured by using a calcein fluorescence technique and the lipidperoxidation products were quantified using the thiobarbituric acid reactive substances (TBARS) method. The cytotoxicity of CM1 was also examined with the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. CM1 was demonstrated to reduce iron-induced redox damage and to decrease the levels of the intracellular iron pool in hepatocytes. CM1 is a potentially useful iron-chelating agent which has potential to ameliorate liver iron overload and ROS-induced lipid peroxidation. CM1 is currently under investigation for oral efficacy.
基金supported by the National Natural Science Funds of China(No.31672413)the National Waterfowl Industrial Technology System(No.CARS-43-6).
文摘In order to explore the role of forkhead box protein O1(FoxO1)in the lipid metabolism and cell proliferation,goose primary hepatocytes were isolated and incubated with insulin or PI3K-Akt-mTOR pathway dual inhibitor NVPBEZ235,and then transfected with FoxO1 interference plasmid.The related parameters of lipid metabolism and cell proliferation were measured.The results firstly showed that FoxO1 interference increased the intracellular TG and lipids concentration(P<0.05);and increased the proliferative index(PI),cell DNA synthesis,protein expression of Cyclin D1 in goose primary hepatocytes(P<0.05).Secondly,the co-treatment of insulin and FoxO1 interference increased the mRNA level and protein content of Cyclin D1(P<0.05);however,there was no significant difference between the insulin treatment and the co-treatment of insulin and miR-FoxO1 interference in the intracellular TG and lipids concentration and PI(P>0.05).Lastly,the decrease of intracellular TG and lipids concentration and PI induced by NVP-BEZ235 was up-regulated by FoxO1 interference significantly(P<0.05).In summary,FoxO1 could regulate the lipids metabolism and cell proliferation mediated by PI3K-Akt-mTOR signaling pathway in goose primary hepatocytes.Further investigations are required to highlight the potential role of FoxO1 in the lipid metabolism and cell proliferation mediated by insulin in goose primary hepatocyte.