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1.
Adv Exp Med Biol ; 1163: 279-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31707708

RESUMO

Allostery is a basic principle that enables proteins to process and transmit cellular information. Protein kinases evolved allosteric mechanisms to transduce cellular signals to downstream signalling components or effector molecules. Protein kinases catalyse the transfer of the terminal phosphate from ATP to protein substrates upon specific stimuli. Protein kinases are targets for the development of small molecule inhibitors for the treatment of human diseases. Drug development has focussed on ATP-binding site, while there is increase interest in the development of drugs targeting alternative sites, i.e. allosteric sites. Here, we review the mechanism of regulation of protein kinases, which often involve the allosteric modulation of the ATP-binding site, enhancing or inhibiting activity. We exemplify the molecular mechanism of allostery in protein kinases downstream of PI3-kinase signalling with a focus on phosphoinositide-dependent protein kinase 1 (PDK1), a model kinase where small compounds can allosterically modulate the conformation of the kinase bidirectionally.


Assuntos
Desenvolvimento de Medicamentos , Fosfatidilinositol 3-Quinases , Proteínas Quinases , Transdução de Sinais , Regulação Alostérica , Sítio Alostérico , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
Acta Cir Bras ; 34(8): e201900802, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618402

RESUMO

PURPOSE: To reveal the function of miR-134 in myocardial ischemia. METHODS: Real-time PCR and western blotting were performed to measure the expression of miR-134, nitric oxide synthase 3 (NOS3) and apoptotic-associated proteins. Lactic dehydrogenase (LDH) assay, cell counting kit-8 (CCK-8), Hoechst 33342/PI double staining and flow cytometry assay were implemented in H9c2 cells, respectively. MiR-134 mimic/inhibitor was used to regulate miR-134 expression. Bioinformatic analysis and luciferase reporter assay were utilized to identify the interrelation between miR-134 and NOS3. Rescue experiments exhibited the role of NOS3. The involvement of PI3K/AKT was assessed by western blot analysis. RESULTS: MiR-134 was high regulated in the myocardial ischemia model, and miR-134 mimic/inhibitor transfection accelerated/impaired the speed of cell apoptosis and attenuated/exerted the cell proliferative prosperity induced by H/R regulating active status of PI3K/AKT signaling. LDH activity was also changed due to the different treatments. Moreover, miR-134 could target NOS3 directly and simultaneously attenuated the expression of NOS3. Co-transfection miR-134 inhibitor and pcDNA3.1-NOS3 highlighted the inhibitory effects of miR-134 on myocardial H/R injury. CONCLUSION: This present work puts insights into the crucial effects of the miR-134/NOS3 axis in myocardial H/R injury, delivering a potential therapeutic technology in future.


Assuntos
Hipóxia/metabolismo , MicroRNAs/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proliferação de Células/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/uso terapêutico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos
3.
J Cancer Res Clin Oncol ; 145(12): 2921-2936, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31620898

RESUMO

PURPOSE: The present study aims to determine whether co-targeting PI3K/Akt and MAPK/ERK pathways in human hypopharyngeal squamous cell carcinoma (HSCC) is a potential anticancer strategy. METHODS: We retrospectively analyzed the clinical data of HSCC patients, and the phosphorylation status of Akt and Erk in HSCC and tumor adjacent tissues was evaluated by immunohistochemistry. MTT and colony formation assay were performed to determine the anti-proliferative effect of PI3K/mTOR inhibitor GDC-0980 and MEK inhibitor Refametinib on HSCC cell line Fadu. Wound-healing and Transwell migration assay were used to analyze the anti-migrative capability of the two drugs. The involved anti-tumor mechanism was explored by flow cytometry, qRT-PCR and western blot. The combinational anticancer effect of GDC-0980 and Refametinib was evaluated according to Chou and Talalay's method. RESULTS: The levels of p-Akt and p-Erk were increased significantly with the progression of clinical stage of HSCC, suggesting PI3K/Akt and MAPK/ERK pathways might be associated with HSCC occurrence and progression. Furthermore, both GDC-0980 and Refametinib showed obvious antitumor effects on FaDu cells. Treatment by the two drugs arrested FaDu cell cycle progression in G1 phase, with reduction of cyclin D1 and p-Rb, in contrast to enhancement of p27. GDC-0980 inhibited FaDu cell migration and reduced metastasis related proteins including p-PKCζ, p-Integrin ß1 and uPA. Combination use of GDC-0980 and Refametinib exhibited strong synergistic anti-tumor effect. CONCLUSION: Dual inhibition of PI3K/Akt and MAPK/ERK pathway by GDC-0980 and Refametinib might be a promising treatment strategy for HSCC patients.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Neoplasias Hipofaríngeas/tratamento farmacológico , Neoplasias Hipofaríngeas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Sinergismo Farmacológico , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Estudos Retrospectivos , Sulfonamidas/farmacologia
4.
Zhongguo Zhong Yao Za Zhi ; 44(16): 3429-3434, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602905

RESUMO

The aim of this paper was to observe the concentration,time and mechanism of autophagy induced by triptolide( TP) in ovarian granulosa cells( OGCs). CCK-8 method was used to compare the inhibitory effects of TP at different concentrations on primary cultured rat OGCs and IC50 was calculated. The effects of TP at different concentrations and time points on the expression of OGCs autophagy factor protein and the cascade of PI3 K/AKT/m TOR pathway were detected by Western blot. The effects of TP,autophagy inducer( brefeldin A) and PI3 K/m TOR inhibitor( NVP-BEZ235) on the expression of PI3 K/AKT/m TOR cascade and autophagy related factor protein were detected by Western blot. The results show that the IC50 of different concentrations of TP on OGCs of rat ovary was14. 65 µmol·L-1,and the minimum inhibitory concentration of TP was 0. 1 µmol·L-1( 100 nmol·L-1). Compared with the control group,the expression levels of beclin1 and LC3Ⅱ in each group were significantly higher than those in the control group( P<0. 05 or P<0. 01). After 12 hours of treatment with TP,brefeldin A and NVP-BEZ235,respectively,compared with the control group,TP could significantly promote the expression level of downstream autophagy effect or molecule beclin1,LC3Ⅱ and inhibit the expression level of LC3Ⅰ,p62 protein( P<0. 05 or P< 0. 01). Moreover,the expression of beclin1 and LC3Ⅱ/LC3Ⅰ in TP group was higher than that in brefeldin A group( P<0. 05 or P<0. 01),and the expression of p62 in TP group was lower than that in brefeldin A group( P<0. 05 or P<0. 01). At the same time,TP could significantly inhibit the expression of p-PI3 K,p-AKT,p-mTOR protein,and the inhibitory effect of TP was better than that of NVP-BEZ235 group. This study suggests that 100 nmol·L-1 TP could induce OGCs autophagy successfully in cultured rat ovary for 12 h; TP may induce OGCs autophagy by inhibiting PI3 k/Akt/m TOR signaling pathway.


Assuntos
Autofagia , Diterpenos/farmacologia , Células da Granulosa/efeitos dos fármacos , Fenantrenos/farmacologia , Transdução de Sinais , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Compostos de Epóxi/farmacologia , Feminino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Serina-Treonina Quinases TOR/metabolismo
5.
Zhongguo Zhong Yao Za Zhi ; 44(17): 3773-3779, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31602952

RESUMO

The aim of this paper was to explore the mechanism of Shenxiong Glucose Injection antagonizing apoptosis of H9 c2 cells induced by H_2O_2. H9 c2 cells were pretreated with 1. 7%,3. 4% and 6. 8% Shenxiong Glucose Injection,and then H_2O_2 was introduced to induce apoptosis in vitro. Cell viability was detected by MTS assay,morphological changes of apoptosis were observed by AO/EB fluorescence staining,apoptosis rate was detected by Annexin/PI method,cell expression profile was detected by gene chip technology,the mRNA of PIK3 CA,Bcl-2,Bax,caspase-3 and GAPDH were detected by qRT-PCR,the protein expression levels of PIK3 CA,AKT,P-AKT,Bcl-2,Bax and caspase-3 were detected by Western blot,and the contents of LDH and MDA were detected by kit. The results showed that Shenxiong Glucose Injection of different concentrations significantly increased the viability of H9 c2 cells treated with H_2O_2( P<0. 01),and reversed H_2O_2-induced apoptosis( P< 0. 01). The microarray experiments showed that 138 genes were altered in H9 c2 cells after treatment with Shenxiong Glucose Injection. The differential expression fold of PIK3 CA associated with PI3 K/AKT pathway was 3. 59. The results of qRT-PCR and Western blot showed that Shenxiong Glucose Injection could down-regulate the mRNA and protein expression levels of caspase-3( P<0. 01),up-regulate the mRNA and protein expression level of PIK3 CA and Bcl-2( P<0. 01),and up-regulate the phosphorylation levels of AKT( P<0. 01) in H_2O_2-treated H9 c2 cells. The protective effect of Shenxiong Glucose Injection on H_2O_2 cells injury was significantly inhibited by LY294002,a PI3 K/AKT pathway inhibitor. The results suggested that Shenxiong Glucose Injection may inhibit H_2O_2-induced H9 c2 cells apoptosis by regulating PI3 K/AKT signaling pathway.


Assuntos
Apoptose , Medicamentos de Ervas Chinesas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Cromonas , Glucose , Morfolinas , Ratos
6.
Anticancer Res ; 39(10): 5297-5310, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570424

RESUMO

BACKGROUND/AIM: Low-molecular weight heparins (LMWHs) may possess putative antitumoral properties; however, the underlying mechanism(s) remains elusive. We evaluated the antiproliferative and antimigratory effects of enoxaparin (a LMWH) in lung adenocarcinoma A549 cells, and assessed the possible mechanism involved, and the effect on doxorubicin's efficacy. MATERIALS AND METHODS: Proliferation and migration were evaluated using BrdU and transwell assays, respectively. Immunoblotting was used to measure PAR-1, PAR-2, MMP-2, ERK1/2 and Akt proteins. Apoptosis and cell cycle studies examined the combined effect of enoxaparin and doxorubicin. RESULTS: Enoxaparin inhibited A549 cell proliferation and migration. Following PAR-1 gene knock down, enoxaparin's effect on A549 cell proliferation was diminished compared to scrambled siRNA. Our experiments verified that enoxaparin-mediated down-regulation of MAPK and PI3K, reduced MMP-2 expression and inhibited A549 cell migration. Additionally, enoxaparin increased doxorubicin's efficacy by enhancing apoptosis, while no effect on cell-cycle progression was observed. CONCLUSION: Results suggest that the anticancer activity of enoxaparin in A549 cells was mediated by the interference of two major PAR-1 downstream signaling pathways, MAPK/ERK and PI3K/Akt, which in turn inhibit proliferation and migration. Therefore, enoxaparin may be promising as an adjunct to traditional chemotherapy for lung cancer and warrants further investigation.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Enoxaparina/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Heparina de Baixo Peso Molecular/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo
7.
Anticancer Res ; 39(10): 5329-5338, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570426

RESUMO

BACKGROUND/AIM: The P13K/Akt signaling pathway is a growth-regulating cellular pathway that is constitutively activated in a variety of human cancers. In previous studies, we reported that a solenopsin analog, compound B (MU-06-SC-608-7), shows inhibitory effects on Akt phosphorylation at a key activation site, as well as on proliferation of tumorigenic cells at sub-micromolar concentrations. The purpose of this study was to evaluate the effect of compound B on downstream effectors of Akt kinase, phosphorylation of Akt at a second activation site, Akt kinase activity in vitro, tumorigenic cell viability and other signaling pathways. MATERIALS AND METHODS: Western blot analyses were performed using WBras1 epithelial and H2009 human carcinoma cells and cell viability assays were performed on H2009 cells. In vitro Akt kinase assays were performed using a commercially available kit. RESULTS: Compound B decreased the phosphorylation of Akt at the Thr308 activation site and key downstream effectors of Akt kinase, but did not directly inhibit Akt kinase. Substantial decreases in cell viability were observed at concentrations above 5 µM. No effect was seen on ERK or JNK pathways. CONCLUSION: The results earmark this compound for further studies as a potential targeted cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos
8.
Chem Biol Interact ; 314: 108843, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31586550

RESUMO

Depression is a common neuropsychiatric disorder whose pathophysiology has been associated with glutamatergic excitotoxicity. Thus, the research for new antidepressant strategies with the ability to mitigate glutamate toxicity has received growing attention. Given this background, the present study sought to investigate the antidepressant-like and neuroprotective effects of Morus nigra (MN) and its major phenolic, syringic acid (SA), against glutamate-induced damage, as well as, the role of the PI3K/Akt/GSK-3ß signaling pathway in these effects. Treatment with MN (3 mg/kg) and SA (1 mg/kg) for 7 days, similar to fluoxetine (10 mg/kg), triggered an antidepressant-like effect. Moreover, the treatments evoked neuroprotection against glutamatergic excitotoxicity in hippocampal slices, and MN treatment also afforded protection in cerebrocortical slices. Notably, ex vivo neuroprotective effect of MN and SA was mediated, at least in part, by PI3K/Akt/GSK-3ß signaling pathway. Furthermore, the ability of MN and SA to counteract the glutamate-induced damage were evaluated in three different in vitro experiments. The hippocampal slices pretreated with MN (0.05 and 0.1 µg/mL) or SA (0.01-0.1 µg/mL) as well as the concomitant treatment with MN (0.01 and 0.05 µg/mL) or SA (0.05 and 0.1 µg/mL) exhibited protection against glutamate toxicity. Interestingly, post-treatment with MN in all doses (0.01-0.1 µg/mL) and SA at dose of 0.1 µg/mL were capable of preventing glutamate-induced cell death. In vitro neuroprotective effect of SA, but not MN, involves the activation of Akt, since the pretreatment with LY294002 completely abolished the protective effect. Overall, MN and SA presented antidepressant-like and neuroprotective effects against glutamatergic excitotoxicity via PI3K/Akt/GSK-3ß.


Assuntos
Antidepressivos/farmacologia , Ácido Gálico/análogos & derivados , Morus/química , Fármacos Neuroprotetores/farmacologia , Fenóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antidepressivos/isolamento & purificação , Comportamento Animal/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Ácido Gálico/isolamento & purificação , Ácido Gálico/farmacologia , Ácido Glutâmico/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Morus/metabolismo , Fármacos Neuroprotetores/isolamento & purificação , Fenóis/isolamento & purificação , Fosfatidilinositol 3-Quinases/metabolismo , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-akt/metabolismo
9.
Cell Physiol Biochem ; 53(4): 656-686, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31573152

RESUMO

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. We have previously reported a discovery of novel anti-melanoma compound 2155-14 (Onwuha-Ekpete et al., J Med Chem. 2014 Feb 27; 57(4):1599-608). In the report presented herein we aim to identify its target(s) and mechanism of action. METHODS: We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Where applicable, one-way analysis of variance (ANOVA) was used followed by Dunnett post hoc test. RESULTS: In the present study, we identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 215514. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. CONCLUSION: Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets.


Assuntos
Apoptose , Autofagia , RNA Helicases DEAD-box/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Avaliação Pré-Clínica de Medicamentos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
BMC Bioinformatics ; 20(1): 507, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31638911

RESUMO

BACKGROUND: Human tumor is a complex tissue with multiple heterogeneous hypoxic regions and significant cell-to-cell variability. Due to the complexity of the disease, the explanation of why anticancer therapies fail cannot be attributed to intrinsic or acquired drug resistance alone. Furthermore, there are inconsistent reports of hypoxia-induced kinase activities in different cancer cell-lines, where increase, decreases, or no change has been observed. Thus, we asked, why are there widely contrasting results in kinase activity under hypoxia in different cancer cell-lines and how does hypoxia play a role in anti-cancer drug sensitivity? RESULTS: We took a modeling approach to address these questions by analyzing the model simulation to explain why hypoxia driven signals can have dissimilar impact on tumor growth and alter the efficacy of anti-cancer drugs. Repeated simulations with varying concentrations of biomolecules followed by decision tree analysis reveal that the highly differential effects among heterogeneous subpopulation of tumor cells could be governed by varying concentrations of just a few key biomolecules. These biomolecules include activated serine/threonine-specific protein kinases (pRAF), mitogen-activated protein kinase kinase (pMEK), protein kinase B (pAkt), or phosphoinositide-4,5-bisphosphate 3-kinase (pPI3K). Additionally, the ratio of activated extracellular signal-regulated kinases (pERK) or pAkt to its respective total was a key factor in determining the sensitivity of pERK or pAkt to hypoxia. CONCLUSION: This work offers a mechanistic insight into how hypoxia can affect the efficacy of anti-cancer drug that targets tumor signaling and provides a framework to identify the types of tumor cells that are either sensitive or resistant to anti-cancer therapy.


Assuntos
Hipóxia/patologia , Neoplasias/patologia , Transdução de Sinais , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases , Modelos Teóricos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinases raf/metabolismo
11.
Life Sci ; 236: 116836, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31493479

RESUMO

AIMS: The present experiment was conceptualised to explore the therapeutic response of tetramethylpyrazine (TMP), a major active constituent of Ligusticum chuanxiong, a Chinese traditional medicinal plant, in high-fat diet (HFD)-streptozotocin (STZ)-induced diabetes in rats and to identify the possible mechanism of action. MAIN METHODS: Dose-reliant effect of oral treatment of TMP (100, 150 and 200 mg/kg/day) for 28 days was evaluated by calculating the alteration in body weight, level of fasting blood glucose (FBG), plasma insulin, homeostasis model assessment (HOMA), serum lipids, oral glucose & intraperitoneal insulin tolerance and glycosylated haemoglobin in HFD-STZ-induced type-2 diabetic (T2D) rats and underlying molecular mechanisms of TMP was also studied. KEY FINDINGS: TMP treatment prominently reduced the level of FBG, glycosylated haemoglobin and revived body weight gain and level of serum insulin dose-dependently in diabetic rats. TMP treatment considerably improved insulin resistance, as observed in oral glucose tolerance and insulin tolerance tests. Moreover, dose-dependent reduction in the level of pro-inflammatory cytokines, C-reactive protein (CRP) and interleukin-6 (IL-6) was observed and their level was found to be significantly reduced in highest dose TMP (200 mg/kg) treated diabetic rats, pointing towards TMP mediated recovery of insulin signalling and a decrease in insulin resistance. The expressions of p-PI3K-p85/p-Akt/GLUT-4 were also significantly up-regulated by TMP (200 mg/kg), suggesting the connection of the PI3K/Akt signal pathway in the anti-hyperglycemic action of TMP. SIGNIFICANCE: These findings suggest that TMP may be used as a potential agent for type-2 diabetes treatment.


Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 2/prevenção & controle , Regulação da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 4/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazinas/farmacologia , Animais , Glicemia , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Transportador de Glucose Tipo 4/genética , Masculino , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Wistar , Transdução de Sinais , Vasodilatadores/farmacologia
12.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 613-618, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537246

RESUMO

Objective To investigate the effect of baicalein (BAI) on autophagy in gastric cancer cell line MGC-803. Methods MGC-803 cells were treated with 0, 5, 15, 25, 50 µmol/L BAI for 24, 48, 72 hours. The proliferation activity of MGC-803 cells was detected by MTT assay. Acridine orange (AO) staining combined with immunofluorescence cytochemical staining was performed to observe the expression of microtubule-associated protein 1 light chain 3 (LC3) and P62 to determine autophagy in MGC-803 cells. The protein levels of LC3, P62, phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), AKT, and p-AKT were detected by Western blot analysis. Results Compared with the control group, BAI significantly inhibited the proliferation of MGC-803 cells in a time- and dose-dependent manner. BAI-treated MGC-803 cells showed a significant increase in acid lysosomes and increased LC3 expression. BAI treatment significantly decreased phosphorylation of PI3K and AKT proteins, increased the ratio of LC3-II/LC3-I and up-regulated the expression of P62 protein. Conclusion Baicalein could inhibit PI3K/AKT signaling pathway and induce autophagy in MGC-803 cells.


Assuntos
Autofagia , Flavanonas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ligação a RNA/metabolismo
13.
Gene ; 721: 144093, 2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31473323

RESUMO

Previous studies have determined that long non-coding RNA (lncRNA) Fer-1-like protein 4 (FER1L4) is suppressed in osteosarcoma (OS) and inhibits the tumorigenesis in a variety of cancer. However, the precise biological of FER1L4 in OS has not been cleared. The aim of this study is to investigate the roles and potential mechanisms of FER1L4 in apoptosis and epithelial-mesenchymal transition (EMT) in OS. In the present study, the levels of FER1L4 were decreased significantly in OS tissues and cell lines compared with non-tumorous tissues or hFOB1.19. Knockdown of FER1L4 in OS cells decreased the apoptosis rate, but increased the OS cell proliferation, upregulated the expression levels of CD133 and Nanog, as well as promoted Twist1 expression, increased the N-cadherin and Vimentin expression. In turn, the opposite trends were observed upon overexpression of FER1L4. In addition, the expression of PI3K, p-AKT (Ser470) and p-AKT (Thr308) was upregulated by siFER1L4, while decreased upon overexpression of FER1L4. MicroRNA (miRNA) -18a-5p, an osteosarcoma-promoting miRNA which was suggested a target of FER1L4 in osteosarcoma, was identified to be a functional target of FER1L4 on the regulating of cell apoptosis and EMT, presently. The effects of FER1L4 overexpression on the markers of cell apoptosis, proliferation, EMT, and stemness and PI3K/AKT signaling were all reversed by miR-18a-5p upregulation. Furthermore, the suppressor of cytokine signaling 5 (SOCS5) was confirmed a target gene of miR-18a-5p by luciferase gene reporter assay and SOCS5 suppression by miR-18a-5p attenuated the effects of FER1L4 overexpression on the OS cells apoptosis and the expressed levels of PI3K, AKT, Twist1, N-cadherin and Vimentin. In conclusion, our data indicated thatthe overexpression of FER1L4 promoted apoptosis and inhibited the EMT markers expression and PI3K/AKT signaling pathway activation in OS cells via downregulating miR-18a-5p to promote SOCS5.


Assuntos
Apoptose , Neoplasias Ósseas/metabolismo , Transição Epitelial-Mesenquimal , MicroRNAs/metabolismo , Osteossarcoma/metabolismo , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Humanos , MicroRNAs/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Proteínas Supressoras da Sinalização de Citocina/genética
14.
Acta Cir Bras ; 34(7): e201900708, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531541

RESUMO

PURPOSE: To investigate the effect of astragaloside IV (As-IV) on myocardial ischemia-reperfusion (I/R) injury in rats and reltaed mechanisms. METHODS: Sixty rats were randomly divided into sham-operated, control I/R and 2.5, 5 and 10 mg/kg As-IV groups, 12 rats in each group. The later three groups were intragastrically administered with As-IV for 7 days, with a dose of 2.5, 5 and 10 mg/kg, respectively. The myocardial I/R injury model was constructed in later four groups. At the end of reperfusion, the cardiac function indexes, serum lactate dehydrogenase (LDH) and creatine kinase (CK) levels, heart weight (HW)/body weight (BW) ratio and infarct size, and expressions of phosphatidylinositol-3 kinase/serine-threonine protein kinase (PI3K/AKT) and glycogen synthase kinase-3ß (GSK-3ß) proteins and the phosphorylated forms (p-AKT, p-GSK-3ß) were determined. RESULTS: Compared with control I/R group, in 5 and 10 mg/kg As-IV groups the left ventricular systolic pressure, fractional shortening and ejection fraction were increased, the left ventricular end-diastolic pressure was decreased, the serum LDH and CK levels were decreased, the HW/BW ratio and myocardial infarct size were decreased, and the p-Akt/Akt ratio and p-GSK-3ß/GSK-3ß ratio were increased (all P < 0.05). CONCLUSION: As-IV can alleviate the myocardial I/R injury in rats through regulating PI3K/AKT/GSK-3ß signaling pathways.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Saponinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Masculino , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Fosforilação , Ratos , Ratos Sprague-Dawley
15.
Life Sci ; 235: 116824, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476305

RESUMO

AIMS: Leflunomide is a disease modifying anti-rheumatic drug (DMARD) beneficial in refractory cases of rheumatoid arthritis. Since leflunomide approval, hepatotoxicity and instructions of liver function monitoring have been recommended. The current work aimed to explore the possible role of inflammation in leflunomide-induced hepatotoxicity with a focus on the TLR4-mediated stimulation of PI3K/mTOR/NFκB pathway. MAIN METHODS: Forty-eight male albino mice were allocated into four different groups (n; 12 mice/group). Group (i): normal mice, Group (ii-iv) mice received escalating dosed/s of leflunomide (2.5, 5 or 10 mg/kg, p.o.) every 48 h for eight weeks. At the end of the study, mice were sacrificed, and blood samples were collected for determination of liver enzymes. Liver samples were collected; (1) formalin-fixed for histologic examination, (2) frozen for PI3K and mTOR genes PCR assays. KEY FINDINGS: Results indicated a significant elevation of liver enzymes in leflunomide-treated mice (10 mg/kg); AST and ALT activities were 218.17 ±â€¯6.83 U/L and 99.83 ±â€¯9.82 U/L versus 130.5 ±â€¯12.79 U/L and 44.72 ±â€¯3.58 U/L in the vehicle group. Additionally, histopathological examination revealed higher necro-inflammatory scores in leflunomide-treated mice. Immunohistochemistry indicated dose-dependent increased staining of TLR4 and caspase 3. Furthermore, leflunomide-treated mice (5 or 10 mg/kg) showed greater staining for NFκB compared to vehicle control. RT-PCR results revealed upregulations in genes expressing PI3K and mTOR by leflunomide. SIGNIFICANCE: The current study highlights the possible role of TLR4-PI3K/mTOR/NFκB in the pathogenesis of leflunomide-induced hepatic injury. A better understanding of mechanisms of leflunomide-induced hepatotoxicity may be of translational implication for the predictive, preventive and therapeutic purposes.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Leflunomida/efeitos adversos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Caspase 3/metabolismo , Relação Dose-Resposta a Droga , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
16.
J Agric Food Chem ; 67(34): 9510-9521, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31382738

RESUMO

Glutamate (Glu) is a critical nutritional regulator of intestinal epithelial homeostasis. In addition, intestinal stem cells (ISCs) at crypt bases are known to play important roles in maintaining the renewal and homeostasis of the intestinal epithelium, and the aspects of communication between Glu and ISCs are still unknown. Here, we identify Glu and mammalian target of rapamycin complex 1 (mTORC1) as essential regulators of ISC expansion. The results showed that extracellular Glu promoted ISC expansion, indicated by increased intestinal organoid forming efficiency and budding efficiency as well as cell proliferation marker Ki67 immunofluorescence and differentiation marker Keratin 20 (KRT20) expression. Moreover, the insulin receptor (IR) mediating phosphorylation of the insulin receptor substrate (IRS) and downstream signaling phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway was involved in this response in ISCs. As expected, Glu-induced mTORC1 signaling activation was observed in the intestinal porcine enterocyte cell line (IPEC-J2), and Glu activated the PI3K/Akt/mTORC1 pathway. Accordingly, PI3K inhibition partially suppressed Glu-induced mTORC1 activation. In addition, Glu increased the phosphorylation levels of IR and IRS, and inhibiting IR downregulated the IRS/PI3K/Akt pathway. Collectively, our findings first indicate that extracellular Glu activates mTORC1 via the IR/IRS/PI3K/Akt pathway and stimulates ISC expansion, providing a new perspective for regulating the growth and health of the intestinal epithelium.


Assuntos
Ácido Glutâmico/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Mucosa Intestinal/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Células-Tronco/metabolismo , Animais , Proteínas Substratos do Receptor de Insulina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Receptor de Insulina/genética , Transdução de Sinais , Suínos
17.
Cell Prolif ; 52(5): e12666, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31407423

RESUMO

OBJECTIVES: Cartilaginous tissue degradation occurs because of the lack of survival of chondrocytes. Here, we ascertained whether bakuchiol (BAK) has the capability of activating chondrocyte proliferation. MATERIALS AND METHODS: The effect of BAK on the proliferation of rat chondrocytes at a concentration of 10 and 20 µmol/L was investigated. The molecular mechanisms involving target binding and signalling pathways were elucidated by RNA-sequencing, qPCR, molecular docking and Western blotting. Matrigel mixed with bakuchiol was implanted locally into rat knee articular cartilage defects to verify the activation of chondrocytes due to bakuchiol in vivo. RESULTS: Bakuchiol implantation resulted in the activation of rat chondrocyte proliferation in a dose-dependent manner. RNA-sequencing revealed 107 differentially expressed genes (DEGs) with 75 that were up-regulated and 32 that were down-regulated, indicating increased activation of the PI3K-Akt and cell cycle pathways. Activation of the phosphorylation of Akt, ERK1/2 and their inhibitors blocked the proliferative effect of bakuchiol treatment, confirming its direct involvement in these signal transduction pathways. Molecular docking and siRNA silencing revealed that estrogen receptor-α (ERα) was the target of bakuchiol in terms of its cell proliferative effect via PI3K activation. Two weeks after implantation of bakuchiol, the appearance and physiological structure of the articular cartilage was more integrated with abundant chondrocytes and cartilage matrix compared to that of the control. CONCLUSIONS: Bakuchiol demonstrated significant bioactivity towards chondrocyte proliferation via the PI3K-Akt and ERK1/2 pathways mediated by estrogen receptor activation and exhibited enhanced promotion of the remodelling of injured cartilage.


Assuntos
Cartilagem Articular/fisiologia , Proliferação de Células/efeitos dos fármacos , Fenóis/farmacologia , Receptores Estrogênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/patologia , Condrócitos/citologia , Condrócitos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos
18.
Life Sci ; 233: 116730, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31390552

RESUMO

AIMS: Dihydroartemisinin (DHA) exhibits potential anticancer activity. However, the biological functions of DHA in prostate cancer remain largely unexplored. In this study, we aim to investigate the anti-proliferative effect and glycolysis regulation of DHA on prostate cancer cell LNCaP. MAIN METHODS: Cell proliferative activity and apoptosis inducing were detected. The gene expression was detected by mRNA microarray and results were analyzed by GO and KEGG pathway database. Expressions of glycolysis key enzymes and PI3K/AKT/HIF-1α were detected by Western blot. KEY FINDINGS: Results indicated that DHA could inhibit the LNCaP cell proliferation considerably and induce cell apoptosis. mRNA microarray showed 1293 genes were upregulated and 2322 genes were downregulated. GO and KEGG enrichment analysis suggested that glycolysis pathway was correlated with DHA inhibited the proliferation on the LNCaP cell. Western blot results showed that DHA can decrease GLUT1 and regulatory enzymes of glycolytic pathway expression probably by suppressing the activity of the intracellular Akt/mTOR and HIF-1 α. SIGNIFICANCE: Experimental validation results indicate that DHA treatment can inhibit the LNCaP cell proliferation and induce apoptosis, which may be related to glycolysis inhibition.


Assuntos
Artemisininas/farmacologia , Biomarcadores Tumorais/metabolismo , Glicólise/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Antimaláricos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas
19.
Int J Nanomedicine ; 14: 5215-5228, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371957

RESUMO

Background: Resveratrol (RSV) has attracted interest as an alternative drug for the treatment of acute lung injury (ALI) and other pulmonary diseases, but its poor oral bioavailability is a limitation. In this study, we employed drug delivery nanotechnology to improve the stability, lung localization and efficacy of orally administered resveratrol to control lung damage leading to ALI. Methods and materials: RSV-loaded lipid-core nanocapsules (RSV-LNCs), prepared by interfacial deposition of biodegradable polymers, were given orally to A/J mice prior to lipopolysaccharide (LPS) intranasal instillation. Inflammatory changes, oxidative stress and lung tissue elastance were assessed 24 h after LPS challenge. Results: RSV-LNCs (5 mg/kg), given 1, 4, 6 or 12 h but not 24 h before provocation, inhibited LPS-induced leukocyte accumulation in the bronchoalveolar fluid (BALF), whereas unloaded nanocapsules (ULNCs) or free RSV (5 mg/kg) were ineffective. RSV-LNCs (2.5-10 mg/kg) but not ULNCs or RSV improved lung function and prevented total leukocyte and neutrophil accumulation equally in both BALF and lung tissue when given 4 h before LPS challenge. Similar findings were seen concerning the generation of a range of pro-inflammatory cytokines such as IL-6, KC, MIP-1α, MIP-2, MCP-1 and RANTES in lung tissue. In addition, only RSV-LNCs inhibited MDA levels and SOD activity in parallel with blockade of the ERK and PI3K/Akt pathways following LPS provocation. Conclusion: Nanoformulation of RSV in biodegradable oil-core polymers is an effective strategy to improve the anti-ALI activity of RSV, suggesting that the modified-release formulation of this plant polyphenol may be of great value in clinical conditions associated with ALI and respiratory failure.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Nanocápsulas/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resveratrol/administração & dosagem , Resveratrol/uso terapêutico , Transdução de Sinais , Lesão Pulmonar Aguda/complicações , Administração Oral , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Masculino , Camundongos Endogâmicos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Hipersensibilidade Respiratória/complicações , Hipersensibilidade Respiratória/patologia , Resveratrol/farmacologia
20.
Sheng Li Xue Bao ; 71(4): 575-580, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31440754

RESUMO

The aim of the present study was to investigate the effect of salidroside (Sal) on inflammatory activation induced by lipopolysaccharide (LPS) in the co-culture of rat alveolar macrophages (AM) NR 8383 and type II alveolar epithelial cells (AEC II) RLE-6TN. CCK-8 colorimetric method was used to detect cell proliferation percentage. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-10 (IL-10) in the supernatant. Western blot was used to examine the expression levels of phosphorylated AKT (p-AKT) and total AKT protein. The results showed that pretreatment of RLE-6TN cells or co-culture of RLE-6TN and NR 8383 cells with 32 and 128 µg/mL Sal for 1 h, followed by continuous culture for 24 h, significantly increased the cell proliferation (P < 0.05). Compared with control group, 32 and 128 µg/mL Sal pretreatment significantly increased the ratio of p-AKT/AKT in RLE-6TN cells (P < 0.05). Pretreatment of 32 µg/mL Sal not only inhibited the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05), but also enhanced the inhibitory effect of RLE-6TN and NR 8383 cells co-culture on the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05). In addition, 32 µg/mL Sal pretreatment promoted LPS-induced IL-10 secretion by NR 8383 cells (P < 0.05), and enhanced the promoting effect of co-culture of RLE-6TN and NR 8383 cells on the IL-10 secretion by LPS-induced NR 8383 cells (P < 0.05). In conclusion, Sal may directly inhibit LPS-induced inflammatory activation of AM (NR 8383), promote the proliferation of AEC II (RLE-6TN) through PI3K/AKT signaling pathway, and enhance the regulatory effect of AEC II on LPS-induced inflammatory activation of AM.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Glucosídeos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Fenóis/farmacologia , Transdução de Sinais , Células Epiteliais Alveolares/metabolismo , Animais , Linhagem Celular , Quimiocina CXCL2/metabolismo , Técnicas de Cocultura , Interleucina-10/metabolismo , Lipopolissacarídeos , Macrófagos Alveolares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismo
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