RESUMO
BACKGROUND & AIMS: Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-ß1 signaling. METHODS: The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice. RESULTS: We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-ß1 (TGF-ß1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-ß1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-ß1-induced fibrogenesis. CONCLUSIONS: Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-ß1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection. IMPACT AND IMPLICATIONS: HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-ß1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection.
Assuntos
Coinfecção , Infecções por HIV , Vírus da Hepatite B , Subunidade alfa do Fator 1 Induzível por Hipóxia , Cirrose Hepática , Transdução de Sinais , Fator de Crescimento Transformador beta1 , Animais , Fator de Crescimento Transformador beta1/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Cirrose Hepática/metabolismo , Cirrose Hepática/virologia , Cirrose Hepática/patologia , Humanos , Infecções por HIV/complicações , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Vírus da Hepatite B/genética , Coinfecção/virologia , Camundongos Endogâmicos C57BL , Hepatite B Crônica/complicações , Hepatite B Crônica/metabolismo , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Proteína gp120 do Envelope de HIV/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , Hepatócitos/patologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/virologia , Modelos Animais de Doenças , Células Hep G2 , MasculinoRESUMO
Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-ß1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-ß1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-ß1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-ß1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-ß1-induced OCT4/Nanog-dependent pathway.
Assuntos
Hepatite B/patologia , Hepatite C/patologia , Cirrose Hepática/patologia , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Actinas/biossíntese , Adulto , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Coinfecção/patologia , Cadeia alfa 1 do Colágeno Tipo I/biossíntese , Feminino , Técnicas de Inativação de Genes , Hepacivirus/metabolismo , Células Estreladas do Fígado/patologia , Células Estreladas do Fígado/virologia , Vírus da Hepatite B/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Fígado/patologia , Cirrose Hepática/virologia , Masculino , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Inibidor Tecidual de Metaloproteinase-1/biossínteseRESUMO
Although the serum-abundant metal-binding protein transferrin (encoded by the Trf gene) is synthesized primarily in the liver, its function in the liver is largely unknown. Here, we generated hepatocyte-specific Trf knockout mice (Trf-LKO), which are viable and fertile but have impaired erythropoiesis and altered iron metabolism. Moreover, feeding Trf-LKO mice a high-iron diet increased their susceptibility to developing ferroptosis-induced liver fibrosis. Importantly, we found that treating Trf-LKO mice with the ferroptosis inhibitor ferrostatin-1 potently rescued liver fibrosis induced by either high dietary iron or carbon tetrachloride (CCl4) injections. In addition, deleting hepatic Slc39a14 expression in Trf-LKO mice significantly reduced hepatic iron accumulation, thereby reducing ferroptosis-mediated liver fibrosis induced by either a high-iron diet or CCl4 injections. Finally, we found that patients with liver cirrhosis have significantly lower levels of serum transferrin and hepatic transferrin, as well as higher levels of hepatic iron and lipid peroxidation, compared with healthy control subjects. Taken together, these data indicate that hepatic transferrin plays a protective role in maintaining liver function, providing a possible therapeutic target for preventing ferroptosis-induced liver fibrosis.
Assuntos
Ferroptose/fisiologia , Ferro/metabolismo , Cirrose Hepática/metabolismo , Fígado/metabolismo , Transferrina/fisiologia , Animais , Intoxicação por Tetracloreto de Carbono/tratamento farmacológico , Intoxicação por Tetracloreto de Carbono/metabolismo , Intoxicação por Tetracloreto de Carbono/patologia , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Cicloexilaminas/farmacologia , Citocinas/análise , Eritropoese/fisiologia , Eritropoetina/análise , Feminino , Ferroptose/efeitos dos fármacos , Hepatócitos/metabolismo , Homeostase , Sobrecarga de Ferro/complicações , Ferro da Dieta/toxicidade , Peroxidação de Lipídeos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/análise , Fenilenodiaminas/farmacologia , Transferrina/análiseRESUMO
Hepatocellular carcinoma (HCC) is a prevalent liver malignancy, which ranks third in the cancer-related cause of deaths in worldwide and ninth in the United States. Currently, HCC is typically diagnosed by ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) scan at its late stage and the survival of HCC patients after diagnosis is usually very poor. Therefore, the development of novel and effective tool for early diagnosis, characterization and staging of HCC patients is of critical importance. Recent studies have demonstrated correlation of HCC with MAGL. In HCC cells, upregulation of MAGL activity enhanced cell invasiveness ability, while pharmacological blockade of MAGL led to significant inhibition of this trend. In this study, we aim to visualize the expression and activity of hepatic MAGL in different HCC cells and HCC patients' samples by taking advantage of positron emission tomography (PET) imaging with our previously developed MAGL radioligand [11C]MAGL-0519. As a result, [11C]MAGL-0519 exhibited higher radioactivity accumulation in HepaG2 and Hepa 1-6 cell lines compared with that of normal liver cells (AML-12 and LX-2), indicating higher MAGL expression levels in these HCC cells. This rationale was then validated by Western blot and immunofluorescent staining analysis. Furthermore, HCC patients' liver sections exhibited significantly increased uptake of [11C]MAGL-0519, which was consistent with the results in cell uptake assays. Taking together, these results provided a biological rationale and built a foundation to use [11C]MAGL-0519 as a potential and effective PET ligand for the diagnosis of HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/patologia , Humanos , Ligantes , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/patologia , Tomografia por Emissão de PósitronsRESUMO
Monoacylglycerol lipase (MAGL) constitutes a serine hydrolase that orchestrates endocannabinoid homeostasis and exerts its function by catalyzing the degradation of 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA). As such, selective inhibition of MAGL represents a potential therapeutic and diagnostic approach to various pathologies including neurodegenerative disorders, metabolic diseases and cancers. Based on a unique 4-piperidinyl azetidine diamide scaffold, we developed a reversible and peripheral-specific radiofluorinated MAGL PET ligand [18F]FEPAD. Pharmacokinetics and binding studies on [18F]FEPAD revealed its outstanding specificity and selectivity towards MAGL in brown adipose tissue (BAT) - a tissue that is known to be metabolically active. We employed [18F]FEPAD in PET studies to assess the abundancy of MAGL in BAT deposits of mice and found a remarkable degree of specific tracer binding in the BAT, which was confirmed by post-mortem tissue analysis. Given the negative regulation of endocannabinoids on the metabolic BAT activity, our study supports the concept that dysregulation of MAGL is likely linked to metabolic disorders. Further, we now provide a suitable imaging tool that allows non-invasive assessment of MAGL in BAT deposits, thereby paving the way for detailed mechanistic studies on the role of BAT in endocannabinoid system (ECS)-related pathologies.
Assuntos
Endocanabinoides , Monoacilglicerol Lipases , Endocanabinoides/metabolismo , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Marrom/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Ligantes , Inibidores Enzimáticos/farmacologiaRESUMO
BACKGROUND AND AIMS: Cholestatic liver disease is characterized by gut dysbiosis and excessive toxic hepatic bile acids (BAs). Modification of gut microbiota and repression of BA synthesis are potential strategies for the treatment of cholestatic liver disease. The purpose of this study was to examine the effects and to understand the mechanisms of the probiotic Lactobacillus rhamnosus GG (LGG) on hepatic BA synthesis, liver injury, and fibrosis in bile duct ligation (BDL) and multidrug resistance protein 2 knockout (Mdr2-/- ) mice. APPROACH AND RESULTS: Global and intestine-specific farnesoid X receptor (FXR) inhibitors were used to dissect the role of FXR. LGG treatment significantly attenuated liver inflammation, injury, and fibrosis with a significant reduction of hepatic BAs in BDL mice. Hepatic concentration of taurine-ß-muricholic acid (T-ßMCA), an FXR antagonist, was markedly increased in BDL mice and reduced in LGG-treated mice, while chenodeoxycholic acid, an FXR agonist, was decreased in BDL mice and normalized in LGG-treated mice. LGG treatment significantly increased the expression of serum and ileum fibroblast growth factor 15 (FGF-15) and subsequently reduced hepatic cholesterol 7α-hydroxylase and BA synthesis in BDL and Mdr2-/- mice. At the molecular level, these changes were reversed by global and intestine-specific FXR inhibitors in BDL mice. In addition, LGG treatment altered gut microbiota, which was associated with increased BA deconjugation and increased fecal and urine BA excretion in both BDL and Mdr2-/- mice. In vitro studies showed that LGG suppressed the inhibitory effect of T-ßMCA on FXR and FGF-19 expression in Caco-2 cells. CONCLUSION: LGG supplementation decreases hepatic BA by increasing intestinal FXR-FGF-15 signaling pathway-mediated suppression of BA de novo synthesis and enhances BA excretion, which prevents excessive BA-induced liver injury and fibrosis in mice.
Assuntos
Ácidos e Sais Biliares , Colestase , Fatores de Crescimento de Fibroblastos/metabolismo , Lacticaseibacillus rhamnosus/metabolismo , Cirrose Hepática , Receptores Citoplasmáticos e Nucleares , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/metabolismo , Ácido Quenodesoxicólico/farmacologia , Colestase/complicações , Colestase/metabolismo , Colestase/terapia , Ácidos Cólicos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Intestinos/microbiologia , Cirrose Hepática/etiologia , Cirrose Hepática/prevenção & controle , Camundongos , Camundongos Knockout , Probióticos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
OBJECTIVES: To enable non-invasive real-time quantification of vasopressin 1A (V1A) receptors in peripheral organs, we sought to develop a suitable PET probe that would allow specific and selective V1A receptor imaging in vitro and in vivo. METHODS: We synthesized a high-affinity and -selectivity ligand, designated compound 17. The target structure was labeled with carbon-11 and tested for its utility as a V1A-targeted PET tracer by cell uptake studies, autoradiography, in vivo PET imaging and ex vivo biodistribution experiments. RESULTS: Compound 17 (PF-184563) and the respective precursor for radiolabeling were synthesized in an overall yield of 49% (over 7 steps) and 40% (over 8 steps), respectively. An inhibitory constant of 0.9 nM towards the V1A receptors was measured, while excellent selectivity over the related V1B, V2 and OT receptor (IC50 >10,000 nM) were obtained. Cell uptake studies revealed considerable V1A binding, which was significantly reduced in the presence of V1A antagonists. Conversely, there was no significant blockade in the presence of V1B and V2 antagonists. In vitro autoradiography and PET imaging studies in rodents indicated specific tracer binding mainly in the liver. Further, the pancreas, spleen and the heart exhibited specific binding of [11C]17 ([11C]PF-184563) by ex vivo biodistribution experiments. CONCLUSION: We have developed the first V1A-targeted PET ligand that is suitable for subtype-selective receptor imaging in peripheral organs including the liver, heart, pancreas and spleen. Our findings suggest that [11C]PF-184563 can be a valuable tool to study the role of V1A receptors in liver diseases, as well as in cardiovascular pathologies.
Assuntos
Benzodiazepinas/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Receptores de Vasopressinas/metabolismo , Triazóis/farmacologia , Animais , Autorradiografia , Benzodiazepinas/farmacocinética , Células CHO , Radioisótopos de Carbono , Cricetulus , Feminino , Ligantes , Fígado/metabolismo , Masculino , Camundongos , Miocárdio/metabolismo , Pâncreas/metabolismo , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/farmacocinética , Ratos Wistar , Baço/metabolismo , Triazóis/farmacocinéticaRESUMO
Alcoholic liver disease (ALD) is associated with gut dysbiosis and hepatic inflammasome activation. While it is known that antimicrobial peptides (AMPs) play a critical role in the regulation of bacterial homeostasis in ALD, the functional role of AMPs in the alcohol-induced inflammasome activation is unclear. The aim of this study was to determine the effects of cathelicidin-related antimicrobial peptide (CRAMP) on inflammasome activation in ALD. CRAMP knockout (Camp-/-) and wild-type (WT) mice were subjected to binge-on-chronic alcohol feeding and synthetic CRAMP peptide was administered. Serum/plasma and hepatic tissue samples from human subjects with alcohol use disorder and/or alcoholic hepatitis were analyzed. CRAMP deficiency exacerbated ALD with enhanced inflammasome activation as shown by elevated serum interleukin (IL)-1ß levels. Although Camp-/- mice had comparable serum endotoxin levels compared to WT mice after alcohol feeding, hepatic lipopolysaccharide (LPS) binding protein (LBP) and cluster of differentiation (CD) 14 were increased. Serum levels of uric acid (UA), a Signal 2 molecule in inflammasome activation, were positively correlated with serum levels of IL-1ß in alcohol use disorder patients with ALD and were increased in Camp-/- mice fed alcohol. In vitro studies showed that CRAMP peptide inhibited LPS binding to macrophages and inflammasome activation stimulated by a combination of LPS and UA. Synthetic CRAMP peptide administration decreased serum UA and IL-1ß concentrations and rescued the liver from alcohol-induced damage in both WT and Camp-/- mice. In summary, CRAMP exhibited a protective role against binge-on-chronic alcohol-induced liver damage via regulation of inflammasome activation by decreasing LPS binding and UA production. CRAMP administration may represent a novel strategy for treating ALD. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Inflamassomos/metabolismo , Hepatopatias Alcoólicas/metabolismo , Fígado/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Biomarcadores/sangue , Disbiose/genética , Disbiose/metabolismo , Disbiose/patologia , Humanos , Inflamassomos/genética , Interleucina-1beta/sangue , Fígado/patologia , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/patologia , Masculino , Camundongos , Camundongos Knockout , Estresse Oxidativo/genética , Ácido Úrico/sangue , CatelicidinasRESUMO
N-methyl-D-aspartate receptors (NMDARs) play critical roles in the physiological function of the mammalian central nervous system (CNS), including learning, memory, and synaptic plasticity, through modulating excitatory neurotransmission. Attributed to etiopathology of various CNS disorders and neurodegenerative diseases, GluN2B is one of the most well-studied subtypes in preclinical and clinical studies on NMDARs. Herein, we report the synthesis and preclinical evaluation of two 11C-labeled GluN2B-selective negative allosteric modulators (NAMs) containing N,N-dimethyl-2-(1H-pyrrolo[3,2-b]pyridin-1-yl)acetamides for positron emission tomography (PET) imaging. Two PET ligands, namely [11C]31 and [11C]37 (also called N2B-1810 and N2B-1903, respectively) were labeled with [11C]CH3I in good radiochemical yields (decay-corrected 28% and 32% relative to starting [11C]CO2, respectively), high radiochemical purity (>99%) and high molar activity (>74 GBq/µmol). In particular, PET ligand [11C]31 demonstrated moderate specific binding to GluN2B subtype by in vitro autoradiography studies. However, because in vivo PET imaging studies showed limited brain uptake of [11C]31 (up to 0.5 SUV), further medicinal chemistry and ADME optimization are necessary for this chemotype attributed to low binding specificity and rapid metabolism in vivo.
Assuntos
Acetamidas/metabolismo , Pirimidinas/metabolismo , Pirróis/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Acetamidas/síntese química , Acetamidas/farmacocinética , Animais , Encéfalo/metabolismo , Radioisótopos de Carbono/química , Feminino , Ligantes , Masculino , Metilação , Camundongos Endogâmicos ICR , Tomografia por Emissão de Pósitrons , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Pirróis/síntese química , Pirróis/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresRESUMO
OBJECTIVE: To explore whether the "sandwich" wound dressing method with a hydrocolloid dressing can effectively reduce surgical site infection rates in sacrococcygeal aseptic operations. METHODS: A retrospective cohort of patients with sacrococcygeal aseptic operations (class I incision category) from January 2017 to March 2018 were divided into intervention (sandwich dressing) and control groups (conventional dressing). The surgical site infections (SSI) rate, wound healing course, hospitalization time, and medical costs in the two groups were determined. To exclude the influence of other factors, operation time, blood loss, age, sex ratio, the distance of the incision from the distal edge to the anus, and initial defecation times were compared between the groups. RESULTS: The SSI rates and medical costs in the interventional group were significantly lower than the control group (0% vs 78.57%, Pâ¯<â¯0.0001; 3.27⯱â¯0.98 vs 5.83⯱â¯1.66 ¥10,000, pâ¯<â¯0.0001). Hospitalization times were also lower in the intervention compared to the control group (17.05⯱â¯4.77 vs 34.50⯱â¯15.47 day, Pâ¯=â¯0.001). CONCLUSIONS: The sandwich wound dressing method with a hydrocolloid dressing can effectively prevent SSI during sacrococcygeal aseptic surgery.
Assuntos
Curativos Hidrocoloides/normas , Região Sacrococcígea/microbiologia , Infecção da Ferida Cirúrgica/prevenção & controle , Adolescente , Adulto , Idoso , Curativos Hidrocoloides/estatística & dados numéricos , Distribuição de Qui-Quadrado , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Região Sacrococcígea/fisiopatologia , Estatísticas não Paramétricas , Infecção da Ferida Cirúrgica/epidemiologiaRESUMO
BACKGROUND & AIMS: The vitronectin receptor integrin αvß3 drives fibrogenic activation of hepatic stellate cells (HSCs). Molecular imaging targeting the integrin αvß3 could provide a non-invasive method for evaluating the expression and the function of the integrin αvß3 on activated HSCs (aHSCs) in the injured liver. In this study, we sought to compare differences in the uptake of [18F]-Alfatide between normal and injured liver to evaluate its utility for assessment of hepatic fibrogenesis. METHODS: PET with [18F]-Alfatide, non-enhanced CT, histopathology, immunofluorescence staining, immunoblotting and gene analysis were performed to evaluate and quantify hepatic integrin αvß3 levels and liver fibrosis progression in mouse models of fibrosis (carbon tetrachloride [CCl4] and bile duct ligation [BDL]). The liver AUC divided by the blood AUC over 30 min was used as an integrin αvß3-PET index to quantify fibrosis progression. Ex vivo analysis of frozen liver tissue from patients with fibrosis and cirrhosis verified the animal findings. RESULTS: Fibrotic mouse livers showed enhanced [18F]-Alfatide uptake and retention compared to control livers. The radiotracer was demonstrated to bind specifically with integrin αvß3, which is mainly expressed on aHSCs. Autoradiography and histopathology confirmed the PET imaging results. Further, the mRNA and protein level of integrin αvß3 and its signaling complex were higher in CCl4 and BDL models than controls. The results obtained from analyses on human fibrotic liver sections supported the animal findings. CONCLUSIONS: Imaging hepatic integrin αvß3 with PET and [18F]-Alfatide offers a potential non-invasive method for monitoring the progression of liver fibrosis. LAY SUMMARY: Integrin αvß3 expression on activated hepatic stellate cells (aHSCs) is associated with HSC proliferation during hepatic fibrogenesis. Herein, we show that a radioactive tracer, [18F]-Alfatide, binds to integrin αvß3 with high affinity and specificity. [18F]-Alfatide could thus be used as a non-invasive imaging biomarker to track hepatic fibrosis progression.
Assuntos
Células Estreladas do Fígado/metabolismo , Integrina alfaVbeta3/metabolismo , Cirrose Hepática , Peptídeos Cíclicos/farmacologia , Tomografia por Emissão de Pósitrons/métodos , Animais , Progressão da Doença , Imunofluorescência/métodos , Radioisótopos de Flúor/farmacologia , Perfilação da Expressão Gênica/métodos , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Imagem Molecular/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Cholesterol 24-hydroxylase, also known as CYP46A1 (EC 1.14.13.98), is a monooxygenase and a member of the cytochrome P450 family. CYP46A1 is specifically expressed in the brain where it controls cholesterol elimination by producing 24S-hydroxylcholesterol (24-HC) as the major metabolite. Modulation of CYP46A1 activity may affect Aß deposition and p-tau accumulation by changing 24-HC formation, which thereafter serves as potential therapeutic pathway for Alzheimer's disease. In this work, we showcase the efficient synthesis and preliminary pharmacokinetic evaluation of a novel cholesterol 24-hydroxylase inhibitor 1 for use in positron emission tomography.
Assuntos
Dióxido de Carbono/química , Isótopos de Carbono , Colesterol 24-Hidroxilase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Compostos Radiofarmacêuticos/síntese química , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Camundongos , Estrutura Molecular , Neuroimagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
Selective DAAO inhibitors have demonstrated promising therapeutic effects in clinical studies, including clinically alleviating symptoms of schizophrenic patients and ameliorating cognitive function in Alzheimer's patients with early phase. Herein we report the synthesis and preliminary evaluation of a 11C-labeled positron emission tomography ligand based on a DAAO inhibitor, DAO-1903 (8). 11C-Isotopologue of 8 was prepared in high radiochemical yield with high radiochemical purity (>99%) and high molar activity (>37 GBq/µmol). In vitro autoradiography studies indicated that the ligand possessed high in vitro specific binding to DAAO, while in vivo dynamic PET studies demonstrated that [11C]8 failed to cross the blood-brain barrier possibly due to moderate brain efflux mechanism. Further chemical scaffold optimization is necessary to overcome limited brain permeability and improve specific binding.
Assuntos
Encéfalo/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Animais , D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Compostos Radiofarmacêuticos/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Transmembrane AMPA receptor regulatory proteins (TARPs) are a recently discovered family of proteins that modulate AMPA receptors activity. Based on a potent and selective TARP subtype γ-8 antagonist, 6-(methyl(4-(pyridin-2-yl)thiazol-2-yl)amino)benzo[d]thiazol-2(3H)-one (compound 9), we perform the radiosynthesis of its 11C-isotopologue 1 and conduct preliminary PET evaluation to test the feasibility of imaging TARP γ-8 dependent receptors in vivo.
Assuntos
Benzoxazóis/química , Encéfalo/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/síntese química , Receptores de AMPA/metabolismo , Animais , Benzoxazóis/síntese química , Radioisótopos de Carbono/química , Estudos de Viabilidade , Marcação por Isótopo , Compostos Radiofarmacêuticos/química , RatosRESUMO
The α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) belong to the family of ionotropic transmembrane receptors for glutamate (iGluRs) that are implicated in the pathology of neurological disorders and neurodegenerative diseases. Inspired by a recently developed positive allosteric modulator of AMPARs, 4-cyclopropyl-7-(3-methoxyphenoxy)-3,4-dihydro-2H-benzo[ e ][1,2,4]thiadiazine 1,1-dioxide (16; EC50 = 2.0 nM), we designed a new synthetic route for N-protected phenolic precursor 13 and efficiently radiolabeled a PET ligand [11C]AMPA-1905 ([11C]16) using a modified one-pot two-step strategy in high radiochemical yield and high molar activity. Preliminary in vivo evaluation was carried out to investigate the suitability of [11C]16 as a potential PET probe for AMPAR imaging.
RESUMO
BACKGROUND: This study aimed to evaluate the association of circular RNA La-related RNA-binding protein 4 (circ-LARP4) with clinical features and prognosis in osteosarcoma patients, and further explore its effect on chemosensitivity in osteosarcoma cells. METHODS: Seventy-two osteosarcoma patients with Enneking stage IIA-IIB who underwent resection were consecutively enrolled, and then, tumor tissues and non-tumor tissues were obtained. Circ-LARP4 in tumor tissue/non-tumor tissue was detected by quantitative polymerase chain reaction. After circ-LARP4 overexpression and negative control overexpression plasmid transfection, relative cell viability (%) was evaluated by Cell Counting Kit-8 in MG63 cells treated by different concentrations of cisplatin, methotrexate, and doxorubicin, and IC50 was calculated. RESULTS: Circ-LARP4 was downregulated in tumor tissue compared with non-tumor tissue and had a good value in distinguishing tumor tissue from non-tumor tissue with an area under curve of 0.829 (95% CI: 0.762-0.859). Meanwhile, tumor circ-LARP4 was negatively correlated with the Enneking stage. After resection, circ-LARP4 high expression patients showed an increased tumor cell necrosis rate to adjuvant chemotherapy compared to circ-LARP4 low expression patients, and circ-LARP4 high expression correlated with prolonged disease-free survival and overall survival. In vitro experiments revealed that circ-LARP4 overexpression elevated the chemosensitivity of MG63 cells to cisplatin and doxorubicin but not methotrexate, with decreased cisplatin IC50 and doxorubicin IC50 concentrations than negative control. Besides, miR-424 overexpression attenuated the chemosensitivity in circ-LARP4 overexpression-treated MG63 cells. CONCLUSION: Circ-LARP4 high expression correlates with decreased Enneking stage and prolonged survival profiles, and it elevates chemosensitivity to cisplatin and doxorubicin via sponging miR-424 in osteosarcoma.
Assuntos
Autoantígenos/genética , Neoplasias Ósseas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/genética , Osteossarcoma/tratamento farmacológico , RNA Circular/genética , Ribonucleoproteínas/genética , Adolescente , Adulto , Antineoplásicos/farmacologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Criança , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Osteossarcoma/genética , Osteossarcoma/mortalidade , Osteossarcoma/patologia , Taxa de Sobrevida , Adulto Jovem , Antígeno SS-BRESUMO
Nonalcoholic fatty liver disease (NAFLD) is a significant public health challenge afflicting approximately 1 billion individuals both in the Western world and in the East world. While liver biopsy is considered as gold standard in the diagnosis and staging of liver fibrosis, noninvasive imaging technologies, including ultrasonography, computed tomography, single-photon emission computed tomography (SPECT), magnetic resonance imaging, and positron emission tomography (PET) could offer more sensitive, comprehensive, and quantitative measurement for NAFLD. In this review, we focus on recent development and applications of PET/SPECT molecular probes that enable multispatial/temporal visualization and quantification of physiopathological progress at the molecular level in the NAFLD. We shall also discuss the limitations of current radioligands and future direction for PET/SPECT probe development.
Assuntos
Sondas Moleculares/análise , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Biomarcadores/metabolismo , Hematologia/métodos , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Tomografia por Emissão de Pósitrons/métodosRESUMO
BACKGROUND: Chronic alcohol intake increases circulating endotoxin levels causing excessive inflammation that aggravates the liver injury. (E)-2,3-dimethoxy-4'-methoxychalcone (L6H21), a derivative of chalcone, has been found to inhibit inflammation in cardiac diseases and nonalcoholic fatty liver disease. However, the use of L6H21 in alcoholic liver disease to inhibit exotoxin-associated inflammation has not been explored. In this study, we examined the effects of L6H21 on EtOH + LPS-induced hepatic inflammation, steatosis, and liver injury and investigated the underlying mechanisms. METHODS: C57BL6 mice were treated with 5% EtOH for 10 days, and LPS was given to the mice 6 hours before sacrificing. One group of mice was supplemented with L6H21 with EtOH and LPS. RAW264.7 cells were used to analyze the effects of L6H21 on macrophage activation. RESULTS: EtOH + LPS treatment significantly increased hepatic steatosis and serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), which were reduced by L6H21 treatment. EtOH + LPS treatment increased hepatic inflammation, as shown by the increased hepatic protein levels of Toll-like receptor-4, p65, and p-IκB, and increased oxidative stress, as shown by protein carbonyl levels and reactive oxygen species formation, which were reduced by L6H21 treatment. In addition, L6H21 treatment markedly inhibited EtOH + LPS-elevated hepatic protein levels of NLRP3, cleaved caspase-1, cleaved IL-1ß, and caspase-1-associated apoptosis. CONCLUSIONS: Our results demonstrate that L6H21 treatment inhibits EtOH + LPS-induced liver steatosis and injury through suppression of NLRP3 inflammasome activation. L6H21 may be used as an alternative strategy for ALD prevention/treatment.
Assuntos
Chalconas/farmacologia , Etanol/efeitos adversos , Inflamassomos/metabolismo , Hepatopatias Alcoólicas/prevenção & controle , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Alanina Transaminase , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/sangue , Caspases/metabolismo , Células Cultivadas , Fígado Gorduroso , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Hepatopatias Alcoólicas/sangue , Hepatopatias Alcoólicas/metabolismo , Masculino , Camundongos , Proteínas dos Microfilamentos/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
BACKGROUND & AIMS: Alcoholic liver disease (ALD) is characterized by gut dysbiosis and increased gut permeability. Hypoxia inducible factor 1α (HIF-1α) has been implicated in transcriptional regulation of intestinal barrier integrity and inflammation. We aimed to test the hypothesis that HIF-1α plays a critical role in gut microbiota homeostasis and the maintenance of intestinal barrier integrity in a mouse model of ALD. METHODS: Wild-type (WT) and intestinal epithelial-specific Hif1a knockout mice (IEhif1α-/-) were pair-fed modified Lieber-DeCarli liquid diet containing 5% (w/v) alcohol or isocaloric maltose dextrin for 24â¯days. Serum levels of alanine aminotransferase and endotoxin were determined. Fecal microbiota were assessed. Liver steatosis and injury, and intestinal barrier integrity were evaluated. RESULTS: Alcohol feeding increased serum levels of alanine aminotransferase and lipopolysaccharide, hepatic triglyceride concentration, and liver injury in the WT mice. These deleterious effects were exaggerated in IEhif1α-/- mice. Alcohol exposure resulted in greater reduction of the expression of intestinal epithelial tight junction proteins, claudin-1 and occludin, in IEhif1α-/- mice. In addition, cathelicidin-related antimicrobial peptide and intestinal trefoil factor were further decreased by alcohol in IEhif1α-/- mice. Metagenomic analysis showed increased gut dysbiosis and significantly decreased Firmicutes/Bacteroidetes ratio in IEhif1α-/- mice compared to the WT mice exposed to alcohol. An increased abundance of Akkermansia and a decreased level of Lactobacillus in IEhif1α-/- mice were also observed. Non-absorbable antibiotic treatment reversed the liver steatosis in both WT and IEhif1α-/- mice. CONCLUSION: Intestinal HIF-1α is essential for the adaptative response to alcohol-induced changes in intestinal microbiota and barrier function associated with elevated endotoxemia and hepatic steatosis and injury. LAY SUMMARY: Alcohol consumption alters gut microbiota and multiple intestinal barrier protecting factors that are regulated by intestinal hypoxia-inducible factor 1α (HIF-1α). Absence of intestinal HIF-1α exacerbates gut leakiness leading to an increased translocation of bacteria and bacterial products to the liver, consequently causing alcoholic liver disease. Intestinal specific upregulation of HIF-1α could be developed as a novel approach for the treatment of alcoholic liver disease.
Assuntos
Disbiose , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Intestinos/microbiologia , Hepatopatias Alcoólicas/etiologia , Animais , Fezes/microbiologia , Hepatite/etiologia , Humanos , Mucosa Intestinal/metabolismo , Masculino , Metagenômica , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: The continuously increasing extracellular matrix stiffness during intervertebral disc degeneration promotes disease progression. In an attempt to obtain novel treatment methods, this study aims to investigate the changes in nucleus pulposus cells under the stimulation of a stiff microenvironment. DESIGN: RNA sequencing and metabolomics experiments were combined to evaluate the primary nucleus pulposus and screen key targets under mechanical biological stimulation. Additionally, small molecules work in vitro were used to confirm the target regulatory effect and investigate the mechanism. In vivo, treatment effects were validated using a rat caudal vertebrae compression model. RESULTS: Our research results revealed that by activating TRPC6, hyperforin, a herbaceous extract can rescue the inflammatory phenotype caused by the stiff microenvironment, hence reducing intervertebral disc degeneration (IDD). Mechanically, it activates mitochondrial fission to inhibit PFKFB3. CONCLUSION: In summary, this study reveals the important bridging role of TRPC6 between mechanical stiffness, metabolism, and inflammation in the context of nucleus pulposus degeneration. TRPC6 activation with hyperforin may become a promising treatment for IDD.