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1.
J Hepatol ; 76(2): 407-419, 2022 02.
Article in English | MEDLINE | ID: mdl-34656650

ABSTRACT

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. The advanced stage of NAFLD, non-alcoholic steatohepatitis (NASH), has been recognized as a leading cause of end-stage liver injury for which there are no FDA-approved therapeutic options. Glutathione S-transferase Mu 2 (GSTM2) is a phase II detoxification enzyme. However, the roles of GSTM2 in NASH have not been elucidated. METHODS: Multiple RNA-seq analyses were used to identify hepatic GSTM2 expression in NASH. In vitro and in vivo gain- or loss-of-function approaches were used to investigate the role and molecular mechanism of GSTM2 in NASH. RESULTS: We identified GSTM2 as a sensitive responder and effective suppressor of NASH progression. GSTM2 was significantly downregulated during NASH progression. Hepatocyte GSTM2 deficiency markedly aggravated insulin resistance, hepatic steatosis, inflammation and fibrosis induced by a high-fat diet and a high-fat/high-cholesterol diet. Mechanistically, GSTM2 sustained MAPK pathway signaling by directly interacting with apoptosis signal-regulating kinase 1 (ASK1). GSTM2 directly bound to the N-terminal region of ASK1 and inhibited ASK1 N-terminal dimerization to subsequently repress ASK1 phosphorylation and the activation of its downstream JNK/p38 signaling pathway under conditions of metabolic dysfunction. CONCLUSIONS: These data demonstrated that hepatocyte GSTM2 is an endogenous suppressor that protects against NASH progression by blocking ASK1 N-terminal dimerization and phosphorylation. Activating GSTM2 holds promise as a therapeutic strategy for NASH. CLINICAL TRIAL NUMBER: IIT-2021-277. LAY SUMMARY: New therapeutic strategies for non-alcoholic steatohepatitis are urgently needed. We identified that the protein GSTM2 exerts a protective effect in response to metabolic stress. Therapies that aim to increase the activity of GSTM2 could hold promise for the treatment of non-alcoholic steatohepatitis.


Subject(s)
Glutathione Transferase/pharmacology , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Non-alcoholic Fatty Liver Disease/prevention & control , Animals , Biopsy/methods , Biopsy/statistics & numerical data , Disease Models, Animal , Gene Targeting/methods , Gene Targeting/standards , Gene Targeting/statistics & numerical data , Glutathione Transferase/metabolism , Hepatocytes/metabolism , Hepatocytes/physiology , Liver/pathology , MAP Kinase Kinase Kinase 5/therapeutic use , Mice , Non-alcoholic Fatty Liver Disease/drug therapy , Sequence Analysis, RNA/methods , Sequence Analysis, RNA/statistics & numerical data
3.
Hepatology ; 74(2): 686-703, 2021 08.
Article in English | MEDLINE | ID: mdl-33576035

ABSTRACT

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease, especially nonalcoholic steatohepatitis (NASH), has become a major cause of liver transplantation and liver-associated death. NASH is the hepatic manifestation of metabolic syndrome and is characterized by hepatic steatosis, inflammation, hepatocellular injury, and different degrees of fibrosis. However, there is no US Food and Drug Administration-approved medication to treat this devastating disease. Therapeutic activators of the AMP-activated protein kinase (AMPK) have been proposed as a potential treatment for metabolic diseases such as NASH. Cordycepin, a natural product isolated from the traditional Chinese medicine Cordyceps militaris, has recently emerged as a promising drug candidate for metabolic diseases. APPROACH AND RESULTS: We evaluated the effects of cordycepin on lipid storage in hepatocytes, inflammation, and fibrosis development in mice with NASH. Cordycepin attenuated lipid accumulation, inflammation, and lipotoxicity in hepatocytes subjected to metabolic stress. In addition, cordycepin treatment significantly and dose-dependently decreased the elevated levels of serum aminotransferases in mice with diet-induced NASH. Furthermore, cordycepin treatment significantly reduced hepatic triglyceride accumulation, inflammatory cell infiltration, and hepatic fibrosis in mice. In vitro and in vivo mechanistic studies revealed that a key mechanism linking the protective effects of cordycepin were AMPK phosphorylation-dependent, as indicated by the finding that treatment with the AMPK inhibitor Compound C abrogated cordycepin-induced hepatoprotection in hepatocytes and mice with NASH. CONCLUSION: Cordycepin exerts significant protective effects against hepatic steatosis, inflammation, liver injury, and fibrosis in mice under metabolic stress through activation of the AMPK signaling pathway. Cordycepin might be an AMPK activator that can be used for the treatment of NASH.


Subject(s)
Deoxyadenosines/pharmacology , Liver Cirrhosis/prevention & control , Liver/drug effects , Non-alcoholic Fatty Liver Disease/drug therapy , AMP-Activated Protein Kinases/metabolism , Animals , Cell Line , Deoxyadenosines/therapeutic use , Hepatocytes , Humans , Liver/immunology , Liver/pathology , Liver Cirrhosis/immunology , Liver Cirrhosis/pathology , Mice , Non-alcoholic Fatty Liver Disease/immunology , Non-alcoholic Fatty Liver Disease/pathology , Signal Transduction/drug effects , Signal Transduction/immunology
4.
Clin Cancer Res ; 27(1): 342-354, 2021 01 01.
Article in English | MEDLINE | ID: mdl-33046519

ABSTRACT

PURPOSE: SHP2 inhibitors offer an appealing and novel approach to inhibit receptor tyrosine kinase (RTK) signaling, which is the oncogenic driver in many tumors or is frequently feedback activated in response to targeted therapies including RTK inhibitors and MAPK inhibitors. We seek to evaluate the efficacy and synergistic mechanisms of combinations with a novel SHP2 inhibitor, TNO155, to inform their clinical development. EXPERIMENTAL DESIGN: The combinations of TNO155 with EGFR inhibitors (EGFRi), BRAFi, KRASG12Ci, CDK4/6i, and anti-programmed cell death-1 (PD-1) antibody were tested in appropriate cancer models in vitro and in vivo, and their effects on downstream signaling were examined. RESULTS: In EGFR-mutant lung cancer models, combination benefit of TNO155 and the EGFRi nazartinib was observed, coincident with sustained ERK inhibition. In BRAFV600E colorectal cancer models, TNO155 synergized with BRAF plus MEK inhibitors by blocking ERK feedback activation by different RTKs. In KRASG12C cancer cells, TNO155 effectively blocked the feedback activation of wild-type KRAS or other RAS isoforms induced by KRASG12Ci and greatly enhanced efficacy. In addition, TNO155 and the CDK4/6 inhibitor ribociclib showed combination benefit in a large panel of lung and colorectal cancer patient-derived xenografts, including those with KRAS mutations. Finally, TNO155 effectively inhibited RAS activation by colony-stimulating factor 1 receptor, which is critical for the maturation of immunosuppressive tumor-associated macrophages, and showed combination activity with anti-PD-1 antibody. CONCLUSIONS: Our findings suggest TNO155 is an effective agent for blocking both tumor-promoting and immune-suppressive RTK signaling in RTK- and MAPK-driven cancers and their tumor microenvironment. Our data provide the rationale for evaluating these combinations clinically.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Immune Checkpoint Inhibitors/pharmacology , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Allosteric Regulation/drug effects , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Drug Synergism , ErbB Receptors/antagonists & inhibitors , Female , Humans , Immune Checkpoint Inhibitors/therapeutic use , Mice , Mutation , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Tumor-Associated Macrophages/drug effects , Tumor-Associated Macrophages/immunology , Xenograft Model Antitumor Assays
5.
Article in English | MEDLINE | ID: mdl-33060070

ABSTRACT

OBJECTIVE: Glucose disposal by insulin-responsive tissues maintains the body glucose homeostasis and insulin resistance leads to a risk of developing type 2 diabetes (T2DM). Insulin stimulates the translocation of glucose transporter isoform 4 (GLUT4) vesicles from intracellular compartments to the plasma membrane to facilitate glucose uptake. However, the underlying mechanisms of GLUT4 vesicle translocation are not well defined. Here we show the role of huntingtin-associated protein 1 (HAP1) in GLUT4 translocation in adipocytes and the pathogenesis of T2DM. RESEARCH DESIGN AND METHODS: The parameters for glucose metabolism including body weight, glucose tolerance and insulin tolerance were assessed in wild-type (WT) and Hap1+/- mice. HAP1 protein expression was verified in adipose tissue. Hap1 mRNA and protein expression was monitored in adipose tissue of high-fat diet (HFD)-induced diabetic mice. Insulin-stimulated GLUT4 vesicle translocation and glucose uptake were detected using immunofluorescence techniques and quantified in primary adipocytes from Hap1-/- mice. The interaction between HAP1 and GLUT4 was assessed by immunofluorescence colocalization and co-immunoprecipitation in HEK293 cells and adipose tissue. The role of sortilin in HAP1 and GLUT4 interaction was approved by co-immunoprecipitation and RNA interference. RESULTS: The expression of Hap1 mRNA and protein was detected in WT mouse adipose tissue and downregulated in adipose tissue of HFD-induced diabetic mice. Hap1+/- mice exhibited increased body weight, pronounced glucose tolerance and significant insulin intolerance compared with the WT mice. HAP1 colocalized with GLUT4 in mouse adipocytes and cotransfected HEK293 cells. Furthermore, the insulin-stimulated GLUT4 vesicle translocation and glucose uptake were defective in Hap1-/- adipocytes. Finally, sortilin mediated the interaction of HAP1 and GLUT4. CONCLUSIONS: Our study showed that HAP1 formed a protein complex with GLUT4 and sortilin, and played a critical role in insulin-stimulated GLUT4 translocation in adipocytes. Its downregulation may contribute to the pathogenesis of diabetes.


Subject(s)
Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 2 , Adipocytes , Animals , Diabetes Mellitus, Type 2/genetics , Glucose Transporter Type 4/genetics , HEK293 Cells , Humans , Mice , Muscle, Skeletal , Protein Isoforms
6.
Cancer Res ; 80(19): 4278-4287, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32747364

ABSTRACT

Advanced ovarian cancers are a leading cause of cancer-related death in women and are currently treated with surgery and chemotherapy. This standard of care is often temporarily successful but exhibits a high rate of relapse, after which, treatment options are few. Here we investigate whether biomarker-guided use of multiple targeted therapies, including small molecules and antibody-drug conjugates, is a viable alternative. A panel of patient-derived ovarian cancer xenografts (PDX), similar in genetics and chemotherapy responsiveness to human tumors, was exposed to 21 monotherapies and combination therapies. Three monotherapies and one combination were found to be active in different subsets of PDX. Analysis of gene expression data identified biomarkers associated with responsiveness to each of the three targeted therapies, none of which directly inhibits an oncogenic driver. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. The distribution of biomarker positivity in The Cancer Genome Atlas data suggests the potential for a similar precision approach in human patients. SIGNIFICANCE: This study exploits a panel of patient-derived xenografts to demonstrate that most ovarian tumors can be matched to effective biomarker-guided treatments.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Biomarkers, Tumor/genetics , Ovarian Neoplasms/drug therapy , Xenograft Model Antitumor Assays/methods , Antineoplastic Agents/pharmacology , Carcinoma, Ovarian Epithelial/drug therapy , Carcinoma, Ovarian Epithelial/genetics , Carcinoma, Ovarian Epithelial/mortality , Carcinoma, Ovarian Epithelial/pathology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Molecular Targeted Therapy/methods , Ovarian Neoplasms/genetics , Ovarian Neoplasms/mortality , Ovarian Neoplasms/pathology , Precision Medicine , Proof of Concept Study
7.
Oncotarget ; 11(4): 443-451, 2020 Jan 28.
Article in English | MEDLINE | ID: mdl-32064048

ABSTRACT

There is a compelling need for new therapeutic strategies for glioblastoma multiforme (GBM). Preclinical target and therapeutic discovery for GBMs is primarily conducted using cell lines grown in serum-containing media, such as U-87 MG, which do not reflect the gene expression profiles of tumors found in GBM patients. To address this lack of representative models, we sought to develop a panel of patient-derived GBM models and characterize their genomic features, using RNA sequencing (RNA-seq) and growth characteristics, both when grown as neurospheres in culture, and grown orthotopically as xenografts in mice. When we compared these with commonly used GBM cell lines in the Cancer Cell Line Encyclopedia (CCLE), we found these patient-derived models to have greater diversity in gene expression and to better correspond to GBMs directly sequenced from patient tumor samples. We also evaluated the potential of these models for targeted therapy, by using the genomic characterization to identify small molecules that inhibit the growth of distinct subsets of GBMs, paving the way for precision medicines for GBM.

8.
Biomed Pharmacother ; 130: 110586, 2020 Oct.
Article in English | MEDLINE | ID: mdl-34321171

ABSTRACT

Sphingosine kinase 1 (SphK1) plays critical roles in the activation of hepatic stellate cells (HSCs) and liver fibrosis. Our previous study found that polydatin ameliorates chronic liver injury and fibrosis by inhibiting oxidative stress. However, whether polydatin exerts an anti-fibrotic effect on liver fibrosis dependent on SphK1 signaling is unknown. We aimed to investigate the role of polydatin in SphK1, which mediates HSC activation and liver fibrosis. C57BL/6 mice were induced using CCl4 5 µL g-1 i.p. twice a week for 6 weeks and treated with or without polydatin. Human immortalized HSC line (LX-2) was induced using platelet-derived growth factor-BB (PDGF-BB) or adenovirus-SphK1 and treated with polydatin. Hepatic macrophage filtration, collagen deposition, expression of α-smooth muscle, active caspase-3, and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells were markedly increased in mice induced by CCl4 for 6 weeks. In contrast, polydatin attenuated collagen synthesis and hepatocyte apoptosis. Furthermore, polydatin exhibited significant anti-proliferative activity against PDGF-BB-induced activated hepatic stellate cells (HSCs). SphK1 was strongly induced in mice exposed to CCl4, whereas its expression and activity were inhibited by polydatin treatment. Finally, SphK1 overexpression in LX-2 cells promoted proliferation of activated HSCs, which could not be reversed by polydatin treatment. These results demonstrate that polydatin attenuates HSC proliferation and activation through inhibition of SphK1 signaling, contributing to the suppression of liver fibrosis.


Subject(s)
Enzyme Inhibitors/pharmacology , Glucosides/pharmacology , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Liver Cirrhosis/metabolism , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Stilbenes/pharmacology , Animals , Apoptosis/drug effects , Biomarkers , Cell Line, Transformed , Cell Proliferation/drug effects , Disease Models, Animal , Humans , Liver Cirrhosis/drug therapy , Liver Cirrhosis/etiology , Liver Cirrhosis/pathology , Mice , Signal Transduction/drug effects
9.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(3): 298-304, 2019 May.
Article in Chinese | MEDLINE | ID: mdl-31631593

ABSTRACT

OBJECTIVE: To determine the effect of Huntingtin-associated protein 1 ( Hap1) on fibroblast proliferation. METHODS: Hap1 knockout ( Hap1 -/-) primary fibroblasts were isolated and cultured in vitro. The proliferation of Hap1 -/- fibroblasts was detected by EdU proliferation assay and cell flow assay. Transcriptome sequencing of the wild-type and Hap1 -/- fibroblasts was screened for proliferation-related genes. Real-time quantitative PCR (qPCR) was performed to verify changes in expressions of related genes. Skin repair was examined in Hap1 knockdown mice with skin wounds. The proliferation of fibroblasts during wound repair was detected by PCNA immunohistochemical staining. RESULTS: Hap1 -/- fibroblasts were successfully cultured. Compared with WT, EdU-positive fibroblasts decreased in Hap1 -/-,with less cells entering the S phase. Transcriptome sequencing of primary fibroblasts identified genes of Cdc25C, E2f7, E2f8 and Ccl5. qPCR confirmed that Hap1 knockout increased E2f7 expression. Hap1 +/- mice had larger skin lesions, slower healing and lower positive density of fibroblast proliferation than those of wild type mice. CONCLUSION: Hap1 may positively regulate fibroblast proliferation by inhibiting the expression of cell cycle negative regulator E2f7.Its deletion inhibits fibroblasts entering the S phase, thereby reducing cell proliferation and affecting wound repair.


Subject(s)
Cell Proliferation , Fibroblasts/cytology , Nerve Tissue Proteins/genetics , Wound Healing , Animals , Cells, Cultured , Gene Knockout Techniques , Mice , Mice, Knockout , Skin/pathology
10.
Exp Cell Res ; 385(1): 111626, 2019 12 01.
Article in English | MEDLINE | ID: mdl-31545977

ABSTRACT

Liver Cholestasis is a widespread disease of broad etiologies and ultimately results in fibrosis, which is still lacking effective therapeutic strategies. Activation of hepatic stellate cells (HSCs) is the key event of liver fibrosis. Here, we aimed to investigate the effect and mechanism of the Slit2 signaling in cholestasis-induced liver fibrosis. Our findings revealed that the serum levels and hepatic expression of Slit2 were significantly increased in patients with primary biliary cirrhosis (PBC). Additionally, Slit2-Tg mice were much more vulnerable to BDL-induced liver injury and fibrosis compared to WT control. Slit2 up-regulation by Slit2 recombinant protein induced proliferation, and inhibited apoptosis of human HSCs cell line LX-2 via p38 and ERK signaling pathway, resulting in the activation of HSCs. In contrast, Slit2 down-regulation by siRNA silencing inhibit the activation of HSCs. In conclusion, Slit2 is involved in the activation of HSCs and liver fibrogenesis, highlighting Slit2 as a potential therapeutic target for liver fibrosis.


Subject(s)
Cholestasis/metabolism , Hepatic Stellate Cells/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Liver Cirrhosis/metabolism , Liver/metabolism , Nerve Tissue Proteins/metabolism , Animals , Apoptosis/physiology , Cell Line , Cell Proliferation/physiology , Cells, Cultured , Down-Regulation/physiology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Signal Transduction/physiology , Up-Regulation/physiology
11.
Gastroenterology ; 156(4): 1156-1172.e6, 2019 03.
Article in English | MEDLINE | ID: mdl-30445007

ABSTRACT

BACKGROUND & AIMS: Although there are associations among oxidative stress, reduced nicotinamide adenine dinucleotide phosphate oxidase (NOX) activation, and hepatocellular carcinoma (HCC) development, it is not clear how NOX contributes to hepatocarcinogenesis. We studied the functions of different NOX proteins in mice after administration of a liver carcinogen. METHODS: Fourteen-day-old Nox1-/- mice, Nox4-/- mice, Nox1-/-Nox4-/- (double-knockout) mice, and wild-type (WT) C57BL/6 mice were given a single intraperitoneal injection of diethylnitrosamine (DEN) and liver tumors were examined at 9 months. We also studied the effects of DEN in mice with disruption of Nox1 specifically in hepatocytes (Nox1ΔHep), hepatic stellate cells (Nox1ΔHep), or macrophages (Nox1ΔMac). Some mice were also given injections of the NOX1-specific inhibitor ML171. To study the acute effects of DEN, 8-12-week-old mice were given a single intraperitoneal injection, and liver and serum were collected at 72 hours. Liver tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and immunoblots. Hepatocytes and macrophages were isolated from WT and knockout mice and analyzed by immunoblots. RESULTS: Nox4-/- mice and WT mice developed liver tumors within 9 months after administration of DEN, whereas Nox1-/- mice developed 80% fewer tumors, which were 50% smaller than those of WT mice. Nox1ΔHep and Nox1ΔHSC mice developed liver tumors of the same number and size as WT mice, whereas Nox1ΔMac developed fewer and smaller tumors, similar to Nox1-/- mice. After DEN injection, levels of tumor necrosis factor, interleukin 6 (IL6), and phosphorylated signal transducer and activator of transcription 3 were increased in livers from WT, but not Nox1-/- or Nox1ΔMac, mice. Conditioned medium from necrotic hepatocytes induced expression of NOX1 in cultured macrophages, followed by expression of tumor necrosis factor, IL6, and other inflammatory cytokines; this medium did not induce expression of IL6 or cytokines in Nox1ΔMac macrophages. WT mice given DEN followed by ML171 developed fewer and smaller liver tumors than mice given DEN followed by vehicle. CONCLUSIONS: In mice given injections of a liver carcinogen (DEN), expression of NOX1 by macrophages promotes hepatic tumorigenesis by inducing the production of inflammatory cytokines. We propose that upon liver injury, damage-associated molecular patterns released from dying hepatocytes activate liver macrophages to produce cytokines that promote tumor development. Strategies to block NOX1 or these cytokines might be developed to slow hepatocellular carcinoma progression.


Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Transformation, Neoplastic/genetics , Hepatitis/genetics , Hepatocytes/pathology , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/pathology , Macrophages/enzymology , NADPH Oxidase 1/genetics , NADPH Oxidase 4/genetics , Alarmins/metabolism , Animals , Carcinoma, Hepatocellular/chemically induced , Cell Proliferation/physiology , Cells, Cultured , Culture Media, Conditioned/pharmacology , Diethylnitrosamine , Enzyme Inhibitors/pharmacology , Hepatic Stellate Cells , Hepatocytes/physiology , Humans , Interleukin-6/metabolism , Liver/metabolism , Liver/pathology , Liver Neoplasms, Experimental/chemically induced , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidase 1/metabolism , Necrosis , STAT3 Transcription Factor/metabolism , Tumor Burden , Tumor Necrosis Factor-alpha/metabolism
12.
Oxid Med Cell Longev ; 2018: 7808656, 2018.
Article in English | MEDLINE | ID: mdl-29743985

ABSTRACT

Liver fibrosis is characterized by activated hepatic stellate cells (HSC) and extracellular matrix accumulation. Blocking the activation of HSC and the inflammation response are two major effective therapeutic strategies for liver fibrosis. In addition to the long history of using andrographolide (Andro) for inflammatory disorders, we aimed at elucidating the pharmacological effects and potential mechanism of Andro on liver fibrosis. In this study, liver fibrosis was induced by carbon tetrachloride (CCl4) and the mice were intraperitoneally injected with Andro for 6 weeks. HSC cell line (LX-2) and primary HSC were also treated with Andro in vitro. Treatment of CCl4-induced mice with Andro decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), Sirius red staining as well as the expression of α smooth muscle actin (α-SMA) and transforming growth factor- (TGF-) ß1. Furthermore, the expression of Toll-like receptor (TLR)4 and NF-κB p50 was also inhibited by Andro. Additionally, in vitro data confirmed that Andro treatment not only attenuated the expression of profibrotic and proinflammatory factors but also blocked the TGF-ß1/Smad2 and TLR4/NF-κB p50 pathways. These results demonstrate that Andro prevents liver inflammation and fibrosis, which is in correlation with the inhibition of the TGF-ß1/Smad2 and TLR4/NF-κB p50 pathways, highlighting Andro as a potential therapeutic strategy for liver fibrosis.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Diterpenes/pharmacology , Liver Cirrhosis/drug therapy , Smad2 Protein/metabolism , Toll-Like Receptor 4/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Liver Cirrhosis/metabolism , Male , Mice , Signal Transduction , Smad2 Protein/antagonists & inhibitors , Toll-Like Receptor 4/antagonists & inhibitors , Transforming Growth Factor beta1/antagonists & inhibitors
13.
Hepatology ; 68(3): 1070-1086, 2018 09.
Article in English | MEDLINE | ID: mdl-29572892

ABSTRACT

Chronic liver disease mediated by activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs) leads to liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the sphingosine kinase (SphK)1-dependent effect on liver fibrosis. The levels of expression and activity of SphK1 were significantly increased in fibrotic livers compared with the normal livers in human. SphK1 was coexpressed with a range of HSC/KC markers including desmin, α-smooth muscle actin (α-SMA) and F4/80 in fibrotic liver. Deficiency of SphK1 (SphK1-/- ) resulted in a marked amelioration of hepatic injury, including transaminase activities, histology, collagen deposition, α-SMA and inflammation, in CCl4 or bile duct ligation (BDL)-induced mice. Likewise, treatment with a specific inhibitor of SphK1, 5C, also significantly prevented liver injury and fibrosis in mice induced by CCl4 or BDL. In cellular levels, inhibition of SphK1 significantly blocked the activation and migration of HSCs and KCs. Moreover, SphK1 knockout in KCs reduced the secretion of CCL2, and SphK1 knockout in HSCs reduced C-C motif chemokine receptor 2 ([CCR2] CCL2 receptor) expression in HSCs. CCL2 in SphK1-/- mice was lower whereas microRNA-19b-3p in SphK1-/- mice was higher compared with wild-type (WT) mice. Furthermore, microRNA-19b-3p downregulated CCR2 in HSCs. The functional effect of SphK1 in HSCs on liver fibrosis was further strengthened by the results of animal experiments using a bone marrow transplantation (BMT) method. CONCLUSION: SphK1 has distinct roles in the activation of KCs and HSCs in liver fibrosis. Mechanistically, SphK1 in KCs mediates CCL2 secretion, and SphK1 in HSCs upregulates CCR2 by downregulation of miR-19b-3p. (Hepatology 2018).


Subject(s)
Liver Cirrhosis/etiology , MicroRNAs/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Receptors, CCR2/metabolism , Animals , Bone Marrow Transplantation , Chemokine CCL2/metabolism , Hepatic Stellate Cells/enzymology , Humans , Kupffer Cells/metabolism , Liver Cirrhosis/metabolism , Mice, Inbred C57BL , Mice, Knockout
15.
Front Neurosci ; 12: 983, 2018.
Article in English | MEDLINE | ID: mdl-30618599

ABSTRACT

Chronic restraint stress (CRS) can affect hypothalamic-pituitary-adrenal (HPA) axis activity and increase glucocorticoid levels. Glucocorticoids are stress hormones that regulate multiple aspects of energy homeostasis. Stress also impairs glucose tolerance. The aim of this study was to investigate the cause of insulin-resistant hyperglycemia during CRS. We produced the CRS models (a 7-day restraint followed by a 3-day free moving procedure, total of 4 cycles for 40 days) in mice, detected the parameters related to glucose metabolism, and compared them to those of the dexamethasone (DEX) injection (0.2 mg/kg i.p., also a 4 cycle procedure as the CRS). The results showed that the CRS induced a moderate (not higher than 11 mmol/L) and irreversible insulin-resistant hyperglycemia in about 1/3 of the individuals, and all the restrained mice had adrenal hypertrophy. CRS induced the apoptosis of neurons in the anterior part of commissural subnucleus of nucleus tractus solitarius (acNTS) in the hyperglycemic mice, and acNTS mechanical damage also led to insulin-resistant hyperglycemia. In contrast, in the DEX-treated mice, adrenal gland atrophy was evident. The glucose and insulin tolerance varied with the delay of determination. DEX exposure in vivo does not induce the apoptosis of neurons in NTS. This study indicates that restraint stress and DEX induce metabolic disorders through different mechanisms. During CRS, injury (apoptosis) of glucose-sensitive acNTS neurons cause dysregulation of blood glucose. This study also suggests the mouse restraint stress model has value as a potential application in the study of stress-induced hyperglycemia.

16.
Exp Cell Res ; 362(2): 343-348, 2018 01 15.
Article in English | MEDLINE | ID: mdl-29196165

ABSTRACT

Activation of hepatic stellate cells (HSCs) is an essential event in the initiation and progression of liver fibrosis. HSCs are believed to be the major source of collagen-producing myofibroblasts in fibrotic livers. A key feature in the pathogenesis of liver fibrosis is fibrillar Collagen I (Col 1) deposition. Osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in HSCs, could drive fibrogenesis by modulating the HSC pro-fibrogenic phenotype and Col 1 expression. Here, we aimed to investigate the molecular mechanism of OPN regulating the activation of HSCs. Our results showed that hepatic expression of OPN was increased in patients with liver fibrosis. In addition, hepatic OPN was positively correlated with Col 1 and α-SMA. Recombinant OPN (rOPN) upregulated Col 1 and α-SMA expression in LX-2 cells. However, OPN knockdown downregulated Col 1 expression. The 3'-UTR of the collagen 1 (Col 1) was identified to bind miR-129-5p. Transfection of miR-129-5p mimic in HSC resulted in a marked reduction of Col 1 expression. Conversely, a decrease in miR-129-5p in HSCs transfected by anti-sense miR-129-5p (AS-miR-129-5p) caused Col 1 upregulation. Furthermore, luciferase reporter assay showed that miR-129-5p directly target the 3'-UTR of Col1α1 mRNA via repressing its post-transcriptional activities. Finally, miR-129-5p level was decreased in fibrotic liver of human, and reduced by rOPN treatment. In contrast, miR-129-5p was induced in HSCs transfected by OPN siRNA. These data suggested that OPN induces Col 1 expression via suppression of miR-129-5p in HSCs.


Subject(s)
Collagen Type I/genetics , Liver Cirrhosis/genetics , MicroRNAs/genetics , Osteopontin/genetics , 3' Untranslated Regions/genetics , Cell Line , Cell Proliferation/genetics , Collagen Type I, alpha 1 Chain , Gene Expression Regulation , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Humans , Liver/metabolism , Liver Cirrhosis/pathology , Osteopontin/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Signal Transduction/genetics
17.
Exp Mol Med ; 49(8): e370, 2017 08 25.
Article in English | MEDLINE | ID: mdl-28857085

ABSTRACT

Chronic high-salt diet-associated renal injury is a key risk factor for the development of hypertension. However, the mechanism by which salt triggers kidney damage is poorly understood. Our study investigated how high salt (HS) intake triggers early renal injury by considering the 'gut-kidney axis'. We fed mice 2% NaCl in drinking water continuously for 8 weeks to induce early renal injury. We found that the 'quantitative' and 'qualitative' levels of the intestinal microflora were significantly altered after chronic HS feeding, which indicated the occurrence of enteric dysbiosis. In addition, intestinal immunological gene expression was impaired in mice with HS intake. Gut permeability elevation and enteric bacterial translocation into the kidney were detected after chronic HS feeding. Gut bacteria depletion by non-absorbable antibiotic administration restored HS loading-induced gut leakiness, renal injury and systolic blood pressure elevation. The fecal microbiota from mice fed chronic HS could independently cause gut leakiness and renal injury. Our current work provides a novel insight into the mechanism of HS-induced renal injury by investigating the role of the intestine with enteric bacteria and gut permeability and clearly illustrates that chronic HS loading elicited renal injury and dysfunction that was dependent on the intestine.


Subject(s)
Acute Kidney Injury/metabolism , Dysbiosis/microbiology , Gastrointestinal Tract/pathology , Sodium Chloride, Dietary/adverse effects , Animals , Bacteroidetes/isolation & purification , Blood Pressure/drug effects , Dysbiosis/etiology , Feces/microbiology , Firmicutes/isolation & purification , Gastrointestinal Microbiome/drug effects , Gastrointestinal Microbiome/genetics , Gene Expression Profiling , Male , Mice , Mice, Inbred C57BL , Permeability/drug effects , RNA/genetics , Time Factors
18.
Nature ; 546(7658): 426-430, 2017 06 15.
Article in English | MEDLINE | ID: mdl-28607489

ABSTRACT

D-type cyclins (D1, D2 and D3) and their associated cyclin-dependent kinases (CDK4 and CDK6) are components of the core cell cycle machinery that drives cell proliferation. Inhibitors of CDK4 and CDK6 are currently being tested in clinical trials for patients with several cancer types, with promising results. Here, using human cancer cells and patient-derived xenografts in mice, we show that the cyclin D3-CDK6 kinase phosphorylates and inhibits the catalytic activity of two key enzymes in the glycolytic pathway, 6-phosphofructokinase and pyruvate kinase M2. This re-directs the glycolytic intermediates into the pentose phosphate (PPP) and serine pathways. Inhibition of cyclin D3-CDK6 in tumour cells reduces flow through the PPP and serine pathways, thereby depleting the antioxidants NADPH and glutathione. This, in turn, increases the levels of reactive oxygen species and causes apoptosis of tumour cells. The pro-survival function of cyclin D-associated kinase operates in tumours expressing high levels of cyclin D3-CDK6 complexes. We propose that measuring the levels of cyclin D3-CDK6 in human cancers might help to identify tumour subsets that undergo cell death and tumour regression upon inhibition of CDK4 and CDK6. Cyclin D3-CDK6, through its ability to link cell cycle and cell metabolism, represents a particularly powerful oncoprotein that affects cancer cells at several levels, and this property can be exploited for anti-cancer therapy.


Subject(s)
Cyclin D3/metabolism , Cyclin-Dependent Kinase 6/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Aminopyridines/pharmacology , Aminopyridines/therapeutic use , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Female , Glycolysis/drug effects , Humans , Mice , Neoplasms/drug therapy , Neoplasms/enzymology , Oxidative Stress/drug effects , Pentose Phosphate Pathway/drug effects , Phosphofructokinase-1/metabolism , Phosphorylation/drug effects , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Purines/pharmacology , Purines/therapeutic use , Pyruvate Kinase/metabolism , Reactive Nitrogen Species/metabolism , Reactive Oxygen Species/metabolism , Serine/metabolism , Xenograft Model Antitumor Assays
19.
Nature ; 535(7610): 148-52, 2016 07 07.
Article in English | MEDLINE | ID: mdl-27362227

ABSTRACT

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS­ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS­ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.


Subject(s)
Neoplasms/drug therapy , Neoplasms/enzymology , Piperidines/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Pyrimidines/pharmacology , Receptor Protein-Tyrosine Kinases/metabolism , Allosteric Regulation/drug effects , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Inhibitory Concentration 50 , MAP Kinase Signaling System/drug effects , Mice , Mice, Nude , Models, Molecular , Neoplasms/pathology , Oncogene Protein p21(ras)/metabolism , Piperidines/chemistry , Piperidines/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Stability/drug effects , Protein Structure, Tertiary/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 11/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Reproducibility of Results , Xenograft Model Antitumor Assays
20.
Mol Cell Endocrinol ; 437: 268-279, 2016 12 05.
Article in English | MEDLINE | ID: mdl-27378149

ABSTRACT

Diabetic nephropathy (DN) is characterized by proliferation of mesangial cells, mesangial hypertrophy and extracellular matrix (ECM) accumulation. Our recent study found that andrographolide inhibited high glucose-induced mesangial cell proliferation and fibronectin expression through inhibition of AP-1 pathway. However, whether andrographolide has reno-protective roles in DN has not been fully elucidated. Here, we studied the pharmacological effects of andrographolide against the progression of DN and high glucose-induced mesangial dysfunction. Diabetes was induced in C57BL/6 mice by intraperitoneal injection of streptozotocin (STZ). After 1 weeks after STZ injection, normal diet was substituted with a high-fat diet (HFD). Diabetic mice were intraperitoneal injected with andrographolide (2 mg/kg, twice a week). After 8 weeks, functional and histological analyses were carried out. Parallel experiments uncovering the molecular mechanism by which andrographolide prevents from DN was performed in mesangial cells. Andrographolide inhibited the increases in fasting blood glucose, triglyceride, kidney/body weight ratio, blood urea nitrogen, serum creatinine and 24-h albuminuria in diabetic mice. Andrographolide also prevented renal hypertrophy and ECM accumulation. Furthermore, andrographolide markedly attenuated NOX1 expression, ROS production and pro-inflammatory cytokines as well. Additionally, andrographolide inhibited Akt/NF-κB signaling pathway. These results demonstrate that andrographolide is protective against the progression of experimental DN by inhibiting renal oxidative stress, inflammation and fibrosis.


Subject(s)
Diabetic Nephropathies/drug therapy , Diterpenes/therapeutic use , Hyperglycemia/drug therapy , Inflammation/pathology , Kidney/pathology , NF-kappa B/metabolism , Oxidative Stress , Proto-Oncogene Proteins c-akt/metabolism , Animals , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , DNA/metabolism , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/pathology , Diabetic Nephropathies/complications , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Diet, High-Fat , Diterpenes/pharmacology , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Glucose/toxicity , Humans , Hyperglycemia/complications , Hypertrophy , Inflammation/complications , Kidney Glomerulus/drug effects , Kidney Glomerulus/pathology , Male , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Mesangial Cells/pathology , Mice, Inbred C57BL , NADP/metabolism , Oxidative Stress/drug effects , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Streptozocin
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