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1.
Adv Exp Med Biol ; 1141: 203-240, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571166

RESUMO

Transporters play an important role in the absorption, distribution, metabolism, and excretion (ADME) of drugs. In recent years, various in vitro, in situ/ex vivo, and in vivo methods have been established for studying transporter function and drug-transporter interaction. In this chapter, the major types of in vitro models for drug transport studies comprise membrane-based assays, cell-based assays (such as primary cell cultures, immortalized cell lines), and transporter-transfected cell lines with single transporters or multiple transporters. In situ/ex vivo models comprise isolated and perfused organs or tissues. In vivo models comprise transporter gene knockout models, natural mutant animal models, and humanized animal models. This chapter would be focused on the methods for the study of drug transporters in vitro, in situ/ex vivo, and in vivo. The applications, advantages, or limitations of each model and emerging technologies are also mentioned in this chapter.


Assuntos
Proteínas de Membrana Transportadoras , Projetos de Pesquisa , Animais , Transporte Biológico , Linhagem Celular , Interações de Medicamentos , Humanos , Técnicas In Vitro , Projetos de Pesquisa/tendências
2.
Adv Exp Med Biol ; 1164: 91-99, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576542

RESUMO

Prostate cancer is the most common male cancer in the USA and the second leading cause of male cancer death in the USA. African American men have higher incidence and mortality rate from prostate cancer compared to Caucasian men in North America, indicating the prostate cancer is a major public health problem in this population. Studies of prostate cancer have been hampered by various factors including (1) restricted access to tissues, (2) difficulties in propagating premalignant lesions and primary prostate tumors in vitro, and (3) limited availability of prostate cell lines for in vitro studies. There is no commercially available pair of non-malignant and tumor cells derived from the same prostate cancer patient. Primary prostate epithelial cells grow for a finite life span and then senesce. Immortalization is defined by continuous growth of otherwise senescing cells and is believed to represent an early stage in tumor progression. To examine these early stages, we have developed in vitro models of prostate epithelial cell immortalization. Generation of human primary epithelial (HPE) cells has been achieved using the serum-free keratinocyte growth medium. Retrovirus containing human telomerase reverse transcriptase (hTERT) was also used for the generation of primary non-malignant and malignant tumor cells. In addition, we have established the first immortalized cell lines of a pair of non-malignant and malignant tumors derived from an African American prostate cancer patient. Interestingly, we have found that the Rock inhibitor and feeder cells induced the conditioned reprogramming (CR) of epithelial cells-normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of viral or cellular genes. More recently, using CR, we have established normal and tumor cultures respectively from a patient prostatectomy. These CR cells grow indefinitely in vitro and retain stable karyology. The tumor-derived CR cells produced tumors in SCID mice. The use of novel pair of non-malignant and malignant tumor cells derived from the same patient provides a unique in vitro model for studies of early prostate cancer and for testing preventive and therapeutic regimens.


Assuntos
Linhagem Celular , Células Epiteliais , Animais , Células Epiteliais/citologia , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata
3.
Adv Exp Med Biol ; 1164: 101-108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576543

RESUMO

Prostate cancer is the most frequently diagnosed solid malignancy in men. Epidemiological studies have shown African-American men to be at higher risk for developing prostate cancer and experience higher death as compared to other ethnic groups. Establishment of prostate cancer cell lines paired with normal cells derived from the same patient is a fundamental breakthrough in cell culture technology and provides a resource to improve our understanding of cancer development and pertinent molecular events. Previous studies have demonstrated that conditional reprogramming (CR) allows the establishment and propagation of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types. Here, we report a new AA prostate cell model, paired normal and cancer epithelial cells from the same patient. "Tumor" cell culture AA-103A was derived from malignant prostate tissues, and "normal" cell culture AA-103B was derived from non-malignant prostate tissues from the prostatectomy specimen of an African-American male. These paired cell cultures have been propagated under CRC conditions to permit direct comparison of the molecular and genetic profiles of the normal epithelium and adenocarcinoma cells for comparison of biomarkers, enabling patient-specific pathological analysis, and molecular and cellular characterization. STR confirmed human origin albeit no karyotypic abnormalities in the two cell lines. Further quantitative PCR analyses demonstrated characteristic markers, including the high level of basal cell marker, the keratin 5 (KRT5) in normal cells and of luminal marker, the androgen receptor (AR) as well as the programmed death-ligand 1 (PD-L1) in tumor cells. Although 3-D sphere formation was observed, the AA-103A of tumor cells did not generate tumors in vivo. We report these paired primary epithelial cultures under CRC growth as a potentially useful tool for studies to understand molecular mechanisms underlying health disparities in prostate cancer.


Assuntos
Afro-Americanos , Linhagem Celular Tumoral , Disparidades nos Níveis de Saúde , Neoplasias da Próstata , Linhagem Celular , Células Epiteliais/citologia , Humanos , Masculino
4.
Adv Exp Med Biol ; 1164: 109-118, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576544

RESUMO

Choosing an appropriate cell model(s) is the first decision to be made before starting a new project or programme of study. Here, we address the rationale that can be behind this decision and we summarize the current cell models that are used to study prostate cancer. Researchers face the challenge of choosing a model that recapitulates the complexity and heterogeneity of prostate cancer. The use of primary prostate epithelial cells cultured from patient tissue is discussed, and the necessity for close clinical-academic collaboration in order to do this is highlighted. Finally, a novel quantitative phase imaging technique is described, along with the potential for cell characterization to not only include gene expression and protein markers but also morphological features, cell behaviour and kinetic activity.


Assuntos
Linhagem Celular Tumoral , Células Epiteliais , Neoplasias da Próstata , Linhagem Celular , Células Epiteliais/citologia , Humanos , Masculino
5.
Anticancer Res ; 39(10): 5515-5524, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570445

RESUMO

BACKGROUND/AIM: Administration of cisplatin in cancer patients is limited by the kidney-related adverse effects; however, a protective strategy is absent. We hypothesized that fucoidan protects the proximal tubule epithelial (TH-1) cells against the effects of cisplatin. MATERIALS AND METHODS: To assess the effect of fucoidan, its effect on reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress response, DNA damage response (DDR), apoptosis, and cell-cycle arrest in TH-1 cells was investigated. RESULTS: Cisplatin increased the accumulation of ROS, leading to excessive ER stress. In presence of cisplatin, treatment of TH-1 cells with fucoidan significantly reduced the ER stress by maintaining the complex of GRP78 with PERK and IRE1α. In particular, fucoidan enhanced the antioxidative capacity through up-regulation of PrPC Furthermore, fucoidan suppressed cisplatin-induced apoptosis and cell-cycle arrest, whereas silencing of PRNP blocked these effects of fucoidan. CONCLUSION: Fucoidan may be a potential adjuvant therapy for cancer patients treated with cisplatin as it preserves renal functionality.


Assuntos
Cisplatino/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Polissacarídeos/farmacologia , Substâncias Protetoras/farmacologia , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Regulação para Cima/efeitos dos fármacos , eIF-2 Quinase/metabolismo
6.
Biol Res ; 52(1): 53, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31542051

RESUMO

BACKGROUND: Oxidative stress is the hallmark of diabetic encephalopathy, which may be caused by hyperglycaemic toxicity. We aimed to discover pharmacologic targets to restore redox homeostasis. We identified the transcription factor Nrf2 as such a target. METHODS: HT22 cells were cultured in 25 or 50 mM D-glucose with various concentrations of sulforaphane (SFN) (from 1.25 to 5.0 µM). Cell viability was tested with the Cell Counting Kit-8 assay. Reactive oxygen species (ROS) production was detected with an inverted fluorescence microscope using the dichlorodihydrofluorescein-diacetate fluorescent probe. The expression of NF-E2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1) and nuclear factor-κB (NF-κB) at the mRNA and protein levels was detected by reverse transcription quantitative polymerase chain reaction and western blotting. RESULT: We found that a high glucose concentration (50 mM) increased the generation of ROS, downregulated the expression of Nrf2/HO-1 and upregulated the expression of NF-κB. Moreover, HT22 cell viability significantly decreased after culture in high-glucose medium for 24, 48 and 72 h, whereas the activation of the Nrf2/HO-1 pathway using a pharmacological Nrf2 activator abrogated this high-glucose-induced toxicity. CONCLUSION: This study suggests that the activation of the Nrf2-ARE signalling pathway might be a therapeutic target for the treatment of diabetic encephalopathy.


Assuntos
Glucose/toxicidade , Hipocampo/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/agonistas , Neuroproteção , Animais , Western Blotting , Linhagem Celular , Eletroforese em Gel de Campo Pulsado , Imunofluorescência , Hipocampo/citologia , Camundongos , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
7.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 606-612, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537245

RESUMO

Objective To investigate the effect of fibroblast growth factor 21 (FGF21) on the lipid accumulation and inflammation induced by palmitate treatment in L02 hepatocytes and the underlying mechanism. Methods L02 cells were infected with lentivirus expressing SIRT1 shRNA to knockdown SIRT1 expression. Wild-type and SIRT1-knockdown L02 cells were treated with 250 mol/L palmitate for 5 days, and then administrated with 1 g/ml FGF21 for 72 hours. Triglycerides in the cells were detected with the infinity triglycerides reagent. Malondialdehyde (MDA) in the cells was assessed by MDA detection assay. Tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) levels in supernatant were measured by ELISA. Reactive oxygen species (ROS) levels were tested by the specific Amplex red ROS detection assay kit from Thermo Fisher Company. The gene expression of SIRT1, peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), superoxide dismutase 2 (SOD2) and catalase (CAT) were measured by real-time quantitative PCR. The protein levels of SIRT1, PGC1α, SOD2 and CAT were detected by Western blot analysis. Mitochondrial membrane potentials were detected by the JC-1staining kit. Mitochondrial oxygen consumption rate (OCR) was detected with the Seahorse XF Mito stress test kit. Results Palmitate increased the triglycerides level, induced the oxidative stress in both the cells and the mitochondria, decreased the gene expression and protein levels of SIRT1, PGC1α, SOD2 and CAT, increased the levels of TNF-α and IL-6, decreased the mitochondrial membrane potential, and impaired the mitochondrial function. FGF21 treatment could attenuate all of these effects caused by palmitate, while SIRT1 knockdown blocked most of the FGF21 effects on the L02 hepatocytes. Conclusion FGF21 activates SIRT1 pathway and inhibites the lipid accumulation, improves the mitochondrial function, and decreases the oxidative stress as well as inflammation in palmitate-treated L02 cells.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Hepatócitos/metabolismo , Inflamação/metabolismo , Sirtuína 1/metabolismo , Catalase/metabolismo , Linhagem Celular , Hepatócitos/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Estresse Oxidativo , Palmitatos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Superóxido Dismutase-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
8.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 625-630, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537248

RESUMO

Objective To investigate the effect of homocysteine (Hcy) on the cardiomyocytes cultured in vitro, and to analyze the role of folic acid in DNA methylation to explore the protective effect and mechanism of folic acid during Hcy exposure of H9C2 cardiomyocytes. Methods H9C2 cells were treated with Hcy at different concentrations (0, 0.5, 1, 2) mmol/L for 24 hours. Cell viability was tested by CCK-8 assay. The apoptosis was detected by flow cytometry. H9C2 cells were divided into 2 mmol/L Hcy group, 0.1 mmol/L folic acid combined with 2 mmol/L Hcy group, 0.1 mmol/L folic acid group and DMSO control group. The above corresponding treatment lasted 24 hours. Then we detected the cell viability and apoptosis. The total DNA methylation level was determined by MethylFlash ELISA kit. DNMT1, DNMT3a, DNMT3b mRNA and protein expression were detected by real-time quantitative PCR and Western blot analysis. Results The number of H9C2 cells treated with different concentrations of Hcy for 24 hours decreased with the increase of Hcy concentration. Compared with the control group, the activity and apoptosis of the cells in the 2 mmol/L Hcy treatment group were reduced, and the number of cells in the folic acid combined with Hcy treatment group was significantly higher than that in the Hcy treatment group. Compared with the other groups, the total apoptosis rate of Hcy treatment group increased, methylation level decreased significantly, and the level of DNA methylation increased in the folic acid combined with Hcy treatment group. The level of DNMT1 mRNA significantly increased only in the folic acid treatment group, and the levels of DNMT1, DNMT3a and DNMT3b were not significantly changed. Conclusion Folic acid can relieve the damage of Hcy to myocardial cells by DNA methylation.


Assuntos
Apoptose , Metilação de DNA , Ácido Fólico/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Homocisteína , Ratos
9.
J Environ Sci (China) ; 85: 94-106, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31471036

RESUMO

Titanium dioxide nanoparticles (TiO2 NPs) are subjected to various transformation processes (chemical, physical and biological processes) in the environment, potentially affecting their bioavailability and toxic properties. However, the size variation of TiO2 NPs during aging process and subsequent effects in mammalian cells are largely unknown. The aim of this study was to illustrate the adverse effects of TiO2 NPs in different sizes (5, 15 and <100 nm) during aging process on human-hamster hybrid (AL) cells. There was an aging-time dependent enhancement of average hydrodynamic size in TiO2 NPs stock suspensions. The cytotoxicity of fresh TiO2 NPs increased in a size-dependent manner; in contrast, their genotoxicity decreased with the increasing sizes of NPs. No significant toxicity difference was observed in cells exposed to either fresh or 60 day-aged TiO2 NPs. Both Fresh and aged TiO2 NPs efficiently induced mitochondrial dysfunction and activated Caspase-3/7 in a size-dependent manner. Using mitochondrial-DNA deficient (ρ0) AL cells, we further discovered that mitochondrial dysfunction made significant contribution to the size-dependent toxicity induced by TiO2 NPs during the aging process. Taken together, our data indicated that TiO2 NPs could significantly induced the cytotoxicity and genotoxicity in an aging time-independent and size-dependent manner, which were triggered by mitochondrial dysfunction. Our study suggested the necessity to include size as an additional parameter for the cautious monitoring of TiO2 NPs disposal before entering the environment.


Assuntos
Nanopartículas/toxicidade , Titânio/toxicidade , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Dano ao DNA , Humanos , Testes de Toxicidade
10.
Arch Virol ; 164(11): 2735-2745, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31486907

RESUMO

Koala retrovirus (KoRV) is unique among endogenous retroviruses because its endogenization is still active. Two major KoRV subtypes, KoRV-A and B, have been described, and KoRV-B is associated with disease and poses a health threat to koalas. Here, we investigated the co-prevalence of KoRV-A and KoRV-B, detected by type-specific PCR and sequencing, and their impact on the health of koalas in three Japanese zoos. We also investigated KoRV proviral loads and found varying amounts of genomic DNA (gDNA) in peripheral blood mononuclear cells (PBMCs). We found that 100% of the koalas examined were infected with KoRV-A and 60% (12/20) were coinfected with KoRV-B. The KoRV-A sequence was highly conserved, whereas the KoRV-B sequence varied among individuals. Interestingly, we observed possible vertical transmission of KoRV-B in one offspring in which the KoRV-B sequence was similar to that of the father but not the mother. Moreover, we characterized the KoRV growth patterns in concanavalin-A-stimulated PBMCs isolated from KoRV-B-coinfected or KoRV-B-uninfected koalas. We quantified the KoRV provirus in gDNA and the KoRV RNA copy numbers in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-seeding. As the study population is housed in captivity, a longitudinal study of these koalas may provide an opportunity to study the transmission mode of KoRV-B. In addition, we characterized KoRV isolates by infecting tupaia cells. The results suggested that tupaia may be used as an infection model for KoRV. Thus, this study may enhance our understanding of KoRV-B coinfection and transmission in the captive koalas.


Assuntos
Retrovirus Endógenos/genética , Gammaretrovirus/patogenicidade , Phascolarctidae/virologia , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/veterinária , Animais , Animais de Zoológico/virologia , Linhagem Celular , Coinfecção/veterinária , Coinfecção/virologia , Retrovirus Endógenos/classificação , Retrovirus Endógenos/isolamento & purificação , Feminino , Gammaretrovirus/classificação , Gammaretrovirus/genética , Gammaretrovirus/isolamento & purificação , Japão/epidemiologia , Masculino , Provírus/genética , Infecções por Retroviridae/virologia , Tupaia/virologia , Carga Viral
11.
Acta Virol ; 63(3): 309-315, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507197

RESUMO

Influenza virus is activated by proteolytic cleavage of hemagglutinin by trypsin. After determining the optimal trypsin concentration, intracellular and extracellular influenza A/PR/8/34 (H1N1) and A/Victoria/361/2011 (H3N2) virus productions were compared in cultures treated with T-705 (favipiravir) and GS 4071 (an active form of oseltamivir). Although both drugs efficiently inhibited extracellular viral RNA release in a dose-dependent manner, T-705 inhibited it to the level of the inoculum without trypsin treatment, while GS 4071 inhibited it to a final level 10 times higher than that without trypsin. T-705 inhibited intracellular viral RNA production to the level of input virus in both trypsin-treated and untreated cells. In contrast, GS 4071 dose-dependently inhibited intracellular viral RNA production in cells treated with trypsin but allowed viral RNA synthesis. The level of maximum inhibition by GS 4071was 10 times higher than that of cells without trypsin and 1,000 times greater than the inoculum titer in cells without trypsin. T-705 inhibited both intracellular and extracellular virus production 1,000 and 10 times more strongly, respectively, than GS 4071. T-705 has powerful anti-influenza activity in the absence of trypsin and even in the trypsin-optimized growth condition, suggesting the therapeutic advantage in treatment of influenza complicated with bacterial pneumonia. Keywords: influenza; T-705; Tamiflu; trypsin; bacterial trypsin-like protease.


Assuntos
Amidas , Antivirais , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Pirazinas , Tripsina , Amidas/farmacologia , Antivirais/farmacologia , Linhagem Celular , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Oseltamivir/farmacologia , Pirazinas/farmacologia , RNA Viral/biossíntese , Tripsina/farmacologia
12.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 41(4): 506-511, 2019 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-31484613

RESUMO

To investigate the expressions of mucosal barrier proteins in colon cell line DLD-1 under hypoxic environment in vitro and its mechanism. Methods After DLD-1 cells were treated separately with hypoxia(l% O2),vitamin D(100 nmol/L),or vitamin D plus hypoxia for 48 hours,the expressions of vitamin D receptor(VDR),tight junction proteins zonula occludens-1(ZO-1),occludin,Claudin-1,and adherent junction protein(E-cadherin)were determined by Western blot.Stable VDR knock-down(Sh-VDR)DLD-1 cell line and control DLD-1 cell line were established by lentivirus package technology and the protein expressions after hypoxia treatment were detected. Results Compared with control group,the expressions of occludin,Claudin-1,and VDR increased significantly after hypoxia treatment(all P<0.001).In addition to the protein expressions of occludin,Claudin-1 and VDR,the expressions of ZO-1 and E-cadherin were also obviously higher in vitamin D plus hypoxia group than in single vitamin D treatment group(all P<0.001).After hypoxia treatment,Sh-VDR cell line showed significantly decreased expressions of ZO-1(P<0.001),occludin(P<0.05),Claudin-1(P<0.01)and E-cadherin(P<0.001)when compared with untreated Sh-VDR cell line. Conclusion VDR acts as a regulator for the expressions of intestinal mucosal barrier proteins under hypoxia environment in DLD-1 colon cell line,indicating that VDR pathway may be another important protective mechanism for gut barrier in low-oxygen environment.


Assuntos
Colo/citologia , Receptores de Calcitriol/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Hipóxia Celular , Linhagem Celular , Claudina-1/metabolismo , Humanos , Ocludina/metabolismo , Junções Íntimas , Vitamina D/farmacologia , Proteína da Zônula de Oclusão-1/metabolismo
13.
Medicine (Baltimore) ; 98(37): e17143, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31517857

RESUMO

The aim of this study was to investigate the alterations of urinary microRNA (miRNA) expression and explore its clinical significance in patients with chronic hepatitis B (CHB).The expression levels of urinary miRNA were detected by miRNA microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) from 106 CHB and 40 healthy controls (Ctrl) subjects. The correlation between the levels of miRNA expression and clinical characteristics were analyzed. Receiver-operator characteristic (ROC) curves were generated to determine the specificity and sensitivity of each individual miRNA. MiRNAs expression were further measured by PCR from exosomes, which were isolated from urine samples. LX2 cells were transfected with miRNA inhibitor and accumulation of cytoplasmic lipid droplets was analyzed by Oil Red O staining.miRNA expression profile analysis showed that 22 miRNAs were upregulated and 55 miRNAs were downregulated in CHB patients compared with Ctrl subjects (fold-change>1.5 and P < .05). miR-92b-3p, miR-770-5p, miR-5196-5p, and miR-7855-5p were significantly higher (P < .0001) in CHB subjects than in Ctrl subjects. ROC curve analysis showed that these four miRNAs were sensitive and specific enough to distinguish CHB and Ctrl subjects. The levels of miR-92b-3p expression were negatively correlated with total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and APOA-1. Moreover, in vitro experiments indicated that inhibition of miR-92b-3p increased lipid droplet formation in LX2 cells.Aberrant expression of miRNAs has been observed in urine of CHB patients. Our findings may provide novel insights into the pathogenesis of CHB and may assist in the diagnosis of patients with CHB.


Assuntos
Hepatite B Crônica/urina , MicroRNAs/urina , Adulto , Biomarcadores/urina , Linhagem Celular , Exossomos/metabolismo , Feminino , Expressão Gênica , Humanos , Gotículas Lipídicas/metabolismo , Masculino , Sensibilidade e Especificidade
14.
Chem Biol Interact ; 311: 108795, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31419397

RESUMO

Citreoviridin (CIT), a mycotoxin and ATP synthase inhibitor, is regarded as one of aetiology factors of cardiac beriberi and Keshan disease. Thiamine (VB1) and selenium (Se) improve the recovery of these two diseases respectively. The underlying mechanisms of cardiotoxic effect of CIT and cardioprotective effect of VB1 and Se have not been fully elucidated. In this study, we found that ectopic ATP synthase was more sensitive to CIT treatment than mitochondrial ATP synthase in H9c2 cardiomyocytes. CIT inhibited the transcriptional activity of peroxisome proliferator activated receptor gamma (PPAR-γ) in mice hearts and H9c2 cells. PPAR-γ agonist attenuated the inhibitory effect of CIT on mechanistic target of rapamycin complex 2 (mTORC2) and stimulatory effect of CIT on autophagy in cardiomyocytes. CIT induced apoptosis through lysosomal-mitochondrial axis in cardiomyocytes. PPAR-γ agonist and autophagy inhibitor alleviated CIT-induced apoptosis and accelerated cardiac biomarker. VB1 and Se accelerated the basal transcriptional activity of PPAR-γ in mice hearts and H9c2 cells. Furthermore, VB1 and Se reversed the effect of CIT on PPAR-γ, autophagy and apoptosis. Our findings defined PPAR-γ-mTORC2-autophagy pathway as the key link between CIT cardiotoxicity and cardioprotective effect of VB1 and Se. The present study would shed new light on the pathogenesis of cardiomyopathy and the cardioprotective mechanism of micronutrients.


Assuntos
Apoptose/efeitos dos fármacos , Aurovertinas/farmacologia , Autofagia/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Selênio/farmacologia , Tiamina/farmacologia , Animais , Aquaporinas/genética , Aquaporinas/metabolismo , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Masculino , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Miocárdio/metabolismo , Miocárdio/patologia , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Proteína X Associada a bcl-2/metabolismo
15.
Chem Biol Interact ; 311: 108794, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31421115

RESUMO

Acanthoic acid (AA) is a pimaradiene diterpene isolated from Acanthopanax koreanum Nakai (Araliaceae), with anti-inflammatory and hepatic-protective effects. The present study intended to reveal the effect and mechanism of AA on nonalcoholic fatty liver disease (NAFLD) associated with lipid accumulation by activating Farnesoid X receptor (FXR) and liver X receptors (LXRs) signaling. C57BL/6 mice were received a modified Lieber-DeCarli diet with 71% high-fat (L-D) and treated with AA (20 and 40 mg/kg) or equal volume of saline for 12 weeks. The regulation of AA on lipid accumulation was also detected in pro-steatotic stimulated AML12 cells with palmitic acid (PA). When L-D diet-fed mice were treated with AA, loss in body weight, liver index, and liver lipid droplet were observed along with reduced triglyceride (TG) and serum transaminase. Furthermore, AA decreased sterol regulatory element binding protein 1 (SREBP-1) and target genes expression, regulated PPARα and PPARγ expressions, ameliorated hepatic fibrosis markers, enhanced hepatic FXR and LXR, and regulated AMPK-LKB1 and SIRT1 signaling pathway. Moreover, AA attenuated lipid accumulation via FXR and LXR activation in steatotic AML-12 cells, which was confirmed by guggulsterones (FXR antagonist) or GW3965 (LXR agonist). Activation of FXR and LXR signaling caused by AA might increase AMPK-SIRT1 signaling and then contribute to modulating lipid accumulation and fatty acid synthesis, which suggested that activated FXR-LXR axis by AA represented an effective strategy for relieving NAFLD.


Assuntos
Diterpenos/farmacologia , Lipogênese/efeitos dos fármacos , Receptores X do Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Dieta Hiperlipídica , Diterpenos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores X do Fígado/agonistas , Receptores X do Fígado/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , PPAR alfa/genética , PPAR alfa/metabolismo , Ácido Palmítico/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/sangue
16.
Cell Physiol Biochem ; 53(2): 355-365, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31385664

RESUMO

BACKGROUND/AIMS: NLRP3 inflammasome activation has been reported to be an early mechanism responsible for glomerular inflammation and injury in obese mice. However, the precise mechanism of obesity-induced NLRP3 inflammasome activation remains unknown. The present study explored whether adipokine visfatin mediates obesity-induced NLRP3 inflammasome activation and consequent podocyte injury. METHODS: Inflammasome formation and immunofluorescence expressions were quantified by confocal microscopy. Caspase-activity, IL-1ß production and VEGF concentrations were measured by ELISA. RESULTS: Confocal microscopic analysis showed that visfatin treatment increased the colocalization of Nlrp3 with Asc or Nlrp3 with caspase-1 in podocytes indicating the formation of NLRP3 inflammasomes. This visfatin-induced NLRP3 inflammasome formation was abolished by pretreatment of podocytes with Asc siRNA. Correspondingly, visfatin treatment significantly increased the caspase-1 activity and IL-1ß production in podocytes, which was significantly attenuated by Asc siRNA transfection. Further RT-PCR and confocal microscopic analysis demonstrated that visfatin treatment significantly decreased the podocin expression (podocyte damage). Podocytes pretreatment with Asc siRNA or caspase-1 inhibitor, WEHD attenuated this visfatin-induced podocin reduction. Furthermore, Asc siRNA transfection was found to preserve podocyte morphology by maintaining the distinct arrangement of F-actin fibers normally lost in response to visfatin. It also prevented podocyte dysfunction by restoring visfatin-induced suppression of VEGF production and secretion. CONCLUSION: Visfatin induces NLRP3 inflammasome activation in podocytes and thereby resulting in podocyte injury.


Assuntos
Adipocinas/imunologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Nicotinamida Fosforribosiltransferase/imunologia , Podócitos/imunologia , Animais , Linhagem Celular , Inflamação/imunologia , Inflamação/patologia , Interleucina-1beta/imunologia , Camundongos , Obesidade/imunologia , Obesidade/patologia , Podócitos/citologia , Podócitos/patologia , Fator A de Crescimento do Endotélio Vascular/imunologia
17.
Cell Physiol Biochem ; 53(2): 366-387, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31385665

RESUMO

BACKGROUND/AIMS: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear. METHODS: To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK. RESULTS: Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. CONCLUSION: These results provide an important role of CK2 in regulating nuclear ERK activities.


Assuntos
Núcleo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular , Caseína Quinase II/metabolismo , Linhagem Celular , Humanos , Carioferinas/metabolismo , Fosforilação , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo
18.
Pestic Biochem Physiol ; 159: 41-50, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400783

RESUMO

Emerging fungal phytodiseases are a food security threat and novel fungicides are in an urgent need. Herein, a series of isobutyrophenone derivatives were designed and synthesized. The derivatives exhibited excellent fungicidal activities against seven fungi. The structure-activity relationship (SAR) study indicated that the introduction of a bromo group at the position 3 or 5 of the phenyl ring, as well as esterification of the 4-hydroxy with a chloroacetyl group, could substantially increase the antifungal activity and spectrum of the compounds. Among all 23 compounds, 2-bromo-3-hydroxy-4-isobutyryl-6-methylphenyl 2-chloroacetate (12b) showed the highest fungicidal activity against all seven tested fungal pathogens with EC50 values ranging from 1.22 to 39.94 µg/mL and exhibited the most potent inhibition against class II fructose-1,6-bisphosphate aldolase with an IC50 of 3.63 µM. The lead compounds were proven to be safe to NIH3T3/293 T cells and silkworm larvae, and relatively stable under different harsh conditions. Detached fruit tests showed the practical potential of lead compounds for fruit (or plant) protection. Taken together, our results indicated that the isobutyrophenone derivatives could be further optimized and developed as advanced leads for new fungicides.


Assuntos
Antifúngicos/química , Antifúngicos/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Animais , Bombyx/metabolismo , Linhagem Celular , Frutose-Bifosfato Aldolase/genética , Humanos , Larva/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Células NIH 3T3 , Relação Estrutura-Atividade
19.
Gene ; 715: 144028, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31374326

RESUMO

BACKGROUND: Type 2 diabetes (T2D) is a complex polygenic disease with unclear mechanism. In an attempt to identify novel genes involved in ß-cell function, we harness a bioinformatics method called Loss-of-function tool (LoFtool) gene score. METHODS: RNA-sequencing data from human islets were used to cross-reference genes within the 1st quartile of most intolerant LoFtool score with the 100th most expressed genes in human islets. Out of these genes, GNAS and EEF1A1 genes were selected for further investigation in diabetic islets, metabolic tissues along with their correlation with diabetic phenotypes. The influence of GNAS and EEF1A1 on insulin secretion and ß-cell function were validated in INS-1 cells. RESULTS: A comparatively higher expression level of GNAS and EEF1A1 was observed in human islets than fat, liver and muscle tissues. Furthermore, diabetic islets displayed a reduced expression of GNAS, but not of EEF1A, compared to non-diabetic islets. The expression of GNAS was positively correlated with insulin secretory index, GLP1R, GIPR and inversely correlated with HbA1c. Diabetic human islets displayed a reduced cAMP generation and insulin secretory capacity in response to glucose. Moreover, siRNA silencing of GNAS in INS-1 cells reduced insulin secretion, insulin content, and cAMP production. In addition, the expression of Insulin, PDX1, and MAFA was significantly down-regulated in GNAS-silenced cells. However, cell viability and apoptosis rate were unaffected. CONCLUSION: LoFtool is a powerful tool to identify genes associated with pancreatic islets dysfunction. GNAS is a crucial gene for the ß-cell insulin secretory capacity.


Assuntos
Cromograninas/biossíntese , Subunidades alfa Gs de Proteínas de Ligação ao GTP/biossíntese , Regulação da Expressão Gênica , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Idoso , Animais , Linhagem Celular , Cromograninas/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Células Secretoras de Insulina/citologia , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Ratos
20.
Gene ; 716: 144031, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31377314

RESUMO

Circular RNAs (circRNAs), a novel class of widespread and diverse endogenous RNAs, have been identified as critical regulators of various cancers, including hepatocellular carcinoma (HCC). However, the specific roles of circRNAs in HCC are largely unknown. In this study, we identified a novel circRNA, circ-IGF1R, in HCC tumour tissues and cell lines. Circ-IGF1R levels were found to be significantly upregulated in HCC tissues compared with levels in paired peritumoural tissues. The high expression levels of circ-IGF1R in HCC were associated with tumour size. Moreover, knocking down circ-IGF1R with siRNA significantly attenuated cell proliferation and induced cell apoptosis and cell cycle arrest in vitro. Further investigation revealed that PI3K/AKT signalling pathway activation was involved in the oncogenic functions of circ-IGF1R in HCC. Our study suggests that circ-IGF1R may be a potential target for the prevention and treatment of HCC.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , RNA/metabolismo , Apoptose , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinase/antagonistas & inibidores , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Regulação para Cima
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