RESUMEN
AIMS: Intestinal ischemia reperfusion (II/R) is a common clinical emergency. Ferroptosis is reported to play a role in II/R injury. Our previous studies revealed that corilagin significantly attenuates intestinal ischemia/reperfusion injuries. However, the underlying molecular mechanism is unclear and requires further study. MATERIALS AND METHODS: DAO, GSSG/T-GSH, MDA, and Fe2+ were measured by assay kits, 4-HNE was assessed by IHC, and 15-LOX was measured by ELISA. Mitochondrial damage was observed by TEM and cellular oxidation levels were detected by C11-BODIPY 581/591 and DHE probes. LC3, p62, Beclin1, ACSL4, GPX4, NCOA4, and ferritin expression were examined by WB in vivo and in vitro. IF, co-IF, q-PCR, and constructed NCOA4-knock-down IEC-6 cells were used to evaluate the role of NCOA4 in the effect of corilagin against II/R injury. Temporal and nucleoplasmic variations with or without corilagin were observed by WB. Co-IP and molecular docking were used to investigate the NCOA4-ferritin interaction. KEY FINDINGS: Corilagin attenuated II/R-induced ferroptosis both in vitro and in vivo. Further study revealed that the anti-ferroptosis bioactivity of corilagin might be due to the modulation of iron homeostasis via inhibition of ferritinophagy in an NCOA4-dependent manner. SIGNIFICANCE: Corilagin might be a potential therapeutic agent for II/R-induced tissue injury.
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Ferroptosis , Isquemia Mesentérica , Daño por Reperfusión , Animales , Ratones , Simulación del Acoplamiento Molecular , Daño por Reperfusión/tratamiento farmacológico , Ferritinas , IsquemiaRESUMEN
Targeted therapies using tyrosine kinase inhibitors (TKIs) against epidermal growth factor receptor (EGFR) have improved the outcomes of patients with non-small cell lung cancer (NSCLC). However, due to genetic mutations of EGFR or activation of other oncogenic pathways, cancer cells can develop resistance to TKIs, resulting in usually temporary and reversible therapeutic effects. Therefore, new anticancer agents are urgently needed to treat drug-resistant NSCLC. In this study, we found that acetyltanshinone IIA (ATA) displayed much stronger potency than erlotinib in inhibiting the growth of drug-resistant NSCLC cells and their-derived xenograft tumors. Our analyses revealed that ATA achieved this effect by the following mechanisms. First, ATA could bind p70S6K at its ATP-binding pocket to prevent phosphorylation, and second by increasing the ubiquitination of p70S6K to cause its degradation. Since phosphorylation of S6 ribosome protein (S6RP) by p70S6K can induce protein synthesis at the ribosome, the dramatic reduction of p70S6K after ATA treatment led to great reductions of new protein synthesis on several cell cycle-related proteins including cyclin D3, aurora kinase A, polo-like kinase, cyclin B1, survivin; and reduced the levels of EGFR and MET. In addition, ATA treatment increased the levels of p53 and p21 proteins, which blocked cell cycle progression in the G1/S phase. Taken together, as ATA can effectively block multiple signaling pathways essential for protein synthesis and cell proliferation, ATA can potentially be developed into a multi-target anti-cancer agent to treat TKI-resistant NSCLC.
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Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/patología , Mutación , Fenantrenos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas S6 Ribosómicas 70-kDaRESUMEN
Kinsenoside (KD) exhibits anti-inflammatory and immunosuppressive effects. Dendritic cells (DCs) are critical regulators of the pathologic inflammatory milieu in liver fibrosis (LF). Herein, we explored whether and how KD repressed development of LF via DC regulation and verified the pathway involved in the process. Given our analysis, both KD and adoptive transfer of KD-conditioned DCs conspicuously reduced hepatic histopathological damage, proinflammatory cytokine release and extracellular matrix deposition in CCl4-induced LF mice. Of note, KD restrained the LF-driven rise in CD86, MHC-II, and CCR7 levels and, simultaneously, upregulated PD-L1 expression on DCs specifically, which blocked CD8+T cell activation. Additionally, KD reduced DC glycolysis, maintained DCs immature, accompanied by IL-12 decrease in DCs. Inhibiting DC function by KD disturbed the communication of DCs and HSCs with the expression or secretion of α-SMA and Col-I declined in the liver. Mechanistically, KD suppressed the phosphorylation of PI3K-AKT driven by LF or PI3K agonist, followed by enhanced nuclear transport of FoxO1 and upregulated interaction of FoxO1 with the PD-L1 promoter in DCs. PI3K inhibitor or si-IL-12 acting on DC could relieve LF, HSC activation and diminish the effect of KD. In conclusion, KD suppressed DC maturation with promoted PD-L1 expression via PI3K-AKT-FoxO1 and decreased IL-12 secretion, which blocked activation of CD8+T cells and HSCs, thereby alleviating liver injury and fibro-inflammation in LF.
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Hepatitis , Fosfatidilinositol 3-Quinasas , 4-Butirolactona/análogos & derivados , Animales , Antígeno B7-H1 , Células Dendríticas/metabolismo , Proteína Forkhead Box O1 , Inflamación/tratamiento farmacológico , Interleucina-12 , Ratones , Monosacáridos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Emerging evidence indicates that the enhancement of microglial autophagy inhibits the NLRP3 inflammasome mediated neuroinflammation in Alzheimer's disease (AD). Meanwhile, low density lipoprotein receptor-related protein 1 (LRP1) highly expressed in microglia is able to negatively regulate neuroinflammation and positively regulate autophagy. In addition, we have previously reported that an active lychee seed fraction enriching polyphenol (LSP) exhibits anti-neuroinflammation in Aß-induced BV-2 cells. However, its molecular mechanism of action is still unclear. In this study, we aim to investigate whether LSP inhibits the NLRP3 inflammasome mediated neuroinflammation and clarify its molecular mechanism in Aß-induced BV-2 cells and APP/PS1 mice. The results showed that LSP dose- and time-dependently activated autophagy by increasing the expression of Beclin 1 and LC3II in BV-2 cells, which was regulated by the upregulation of LRP1 and its mediated AMPK signaling pathway. In addition, both the Western blotting and fluorescence microscopic results demonstrated that LSP could significantly suppress the activation of NLRP3 inflammasome by inhibiting the expression of NLRP3, ASC, the cleavage of caspase-1, and the release of IL-1ß in Aß(1-42)-induced BV-2 cells. In addition, the siRNA LRP1 successfully abolished the effect of LSP on the activation of AMPK and its mediated autophagy, as well as the inhibition of NLRP3 inflammasome. Furthermore, LSP rescued PC-12 cells which were induced by the conditioned medium from Aß(1-42)-treated BV-2 cells. Moreover, LSP improved the cognitive function and inhibited the NLRP3 inflammasome in APP/PS1 mice. Taken together, LSP inhibited the NLRP3 inflammasome-mediated neuroinflammation in the in vitro and in vivo models of AD, which was closely associated with the LRP1/AMPK-mediated autophagy. Thus, the findings from this study further provide evidences for LSP serving as a potential drug for the treatment of AD in the future.
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Proteínas Quinasas Activadas por AMP/metabolismo , Péptidos beta-Amiloides , Inflamasomas/antagonistas & inhibidores , Litchi , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos , Polifenoles/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Autofagia/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Inflamasomas/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , ARN Interferente Pequeño , Ratas , SemillasRESUMEN
Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Our previous studies have proven that Trillium tschonoskii Maxim. (TTM), a traditional Chinese medicine, possesses potent anti-tumor effect. However, the detailed components and molecular mechanism of TTM in anti-NSCLC are still unknown. In the present experiment, polyphyllin VI (PPVI) was successfully isolated from TTM with guidance of the anti-proliferative effect in A549 cells, and the cell death of PPVI treated A549 and H1299 cells was closely linked with the increased intracellular ROS levels. In addition, PPVI induced apoptosis by promoting the protein expression of Bax/Bcl2, caspase-3 and caspase-9, and activated autophagy by improving LC3 II conversion and GFP-LC3 puncta formation in A549 and H1299 cells. The mechanism study found that the activity of mTOR which regulates cell growth, proliferation and autophagy was significantly suppressed by PPVI. Accordingly, the PI3K/AKT and MEK/ERK pathways positively regulating mTOR were inhibited, and AMPK negatively regulating mTOR was activated. In addition, the downstream of mTOR, ULK1 at Ser 757 which downregulates autophagy was inhibited by PPVI. The apoptotic cell death induced by PPVI was confirmed, and it was significantly suppressed by the overexpression of AKT, ERK and mTOR, and the induced autophagic cell death which was depended on the Atg7 was decreased by the inhibitors, such as LY294002 (LY), Bafilomycin A1 (Baf), Compound C (CC) and SBI-0206965 (SBI). Furthermore, the mTOR signaling pathway was regulated by the increased ROS as the initial signal in A549 and H1299 cells. Finally, the anti-tumor growth activity of PPVI in vivo was validated in A549 bearing athymic nude mice. Taken together, our data have firstly demonstrated that PPVI is the main component in TTM that exerts the anti-proliferative effect by inducing apoptotic and autophagic cell death in NSCLC via the ROS-triggered mTOR signaling pathway, and PPVI may be a promising candidate for the treatment of NSCLC in future.
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Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Saponinas/farmacología , Saponinas/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Muerte Celular Autofágica/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , TrilliumRESUMEN
Multidrug resistance protein 1 (MRP1/ABCC1) actively transports a variety of drugs, toxic molecules and important physiological substrates across the plasma membrane. It can confer broad-spectrum multidrug resistance and can decrease the bioavailability of many important drugs. Substrates of MRP1 include anti-cancer agents, antibiotics, antivirals, antidepressants and anti-inflammatory drugs. Using calcein as a fluorescent reporter in a high content uptake assay, we recently reported the identification of 12 MRP1 inhibitors after screening an anti-cancer library of 386 compounds. Here, we describe the development of a new high content imaging-based uptake assay using doxorubicin as a fluorescent reporter. Screening the same anti-cancer library of 386 compounds, the new assay identified a total of 28 MRP1 inhibitors including 16 inhibitors that have not been previously reported as inhibitors of MRP1. Inhibition of MRP1 activity was confirmed using flow cytometry and confocal microscopy-based transport assays. Six drugs (afatinib, celecoxib, doramapimod, mifepristone, MK-2206 and rosiglitazone) were evaluated for their ability to reverse resistance of MRP1-overexpressing H69AR lung cancer cells against vincristine, doxorubicin and etoposide. Mifepristone and doramapimod were most effective in reversal of resistance against vincristine while mifepristone and rosiglitazone were most successful in resensitizing H69AR cells against doxorubicin. Furthermore, resistance towards etoposide was completely reversed in the presence of celecoxib or doramapimod. Selected drugs were also evaluated for resistance reversal in HEK cells that overexpress P-glycoprotein or breast cancer resistance protein. Our results indicate mifepristone and doramapimod as pan inhibitors of these three drug transporters while celecoxib exhibited selective MRP1 inhibition. Together, our findings signify the importance of MRP1 in drug discovery and demonstrate the effectiveness and value of doxorubicin-based high content screening approach. Anti-cancer agents that exhibit MRP1 inhibition may be used to reverse multidrug resistance or to improve the efficacy and reduce the toxicity of various cancer chemotherapies. On the other hand, anti-cancer drugs that did not interact with MRP1 carry a low risk for developing MRP1-mediated resistance.
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Antineoplásicos/farmacología , Doxorrubicina/farmacología , Fluoresceínas/química , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/química , Bioensayo , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Células HEK293 , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transporte de Proteínas/efectos de los fármacos , Reproducibilidad de los ResultadosRESUMEN
In the effort to identify natural products that regulate immunity and inflammation, we found that nitidine chloride (NC), an alkaloid from herb Zanthoxylum nitidum, enhanced IL-10 production in lipopolysaccharide (LPS)-stimulated myeloid cells. While NC was shown to be capable of inhibiting topoisomerase I (TOP1), NC analogs that could not inhibit TOP1 failed to increase IL-10 production. Moreover, medicinal TOP1 inhibitors TPT and SN-38 also augmented IL-10 production significantly, whereas knockdown of TOP1 prevented NC, TPT, and SN-38 from enhancing IL-10 expression. Thus, NC promoted IL-10 production by inhibiting TOP1. In LPS-induced endotoxemic mice, NC and TOP1 inhibitors increased IL-10 production, suppressed inflammatory responses, and reduced mortality remarkably. The anti-inflammatory activities of TOP1 inhibition were markedly reduced by IL-10-neutralizing antibody and largely absent in IL-10-deficient mice. In LPS-stimulated RAW264.7 cells and in peritoneal macrophages from endotoxemic mice, NC and TOP1 inhibitors significantly enhanced the activation of Akt, a critical signal transducer for IL-10 production, and inhibition of Akt prevented these compounds from enhancing IL-10 production and ameliorating endotoxemia. These data indicated that NC and TOP1 inhibitors are able to exert anti-inflammatory action through enhancing Akt-mediated IL-10 production and may assist with the treatment of inflammatory diseases.
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Antiinflamatorios/farmacología , Benzofenantridinas/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Interleucina-10/metabolismo , Animales , Línea Celular , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Células THP-1RESUMEN
Ischemia-reperfusion (I/R) injury is accompanied with high morbidity and mortality and has seriously negative social and economic influences. Unfortunately, few effective therapeutic strategies are available to improve its outcome. Berberine is a natural medicine possessing multiple beneficial biological activities. Emerging evidence indicates that berberine has potential protective effects against I/R injury in brain, heart, kidney, liver, intestine and testis. However, up-to-date review focusing on the beneficial role of berberine against I/R injury is not yet available. In this paper, results from animal models and clinical studies are concisely presented and its mechanisms are discussed. We found that berberine ameliorates I/R injury in animal models via its anti-oxidant, anti-apoptotic and anti-inflammatory effects. Moreover, berberine also attenuates I/R injury by suppressing endoplasmic reticulum stress and promoting autophagy. Additionally, regulation of periphery immune system may also contributes to the beneficial effect of berberine against I/R injury. Although clinical evidence is limited, the current studies indicate that berberine may attenuate I/R injury via inhibiting excessive inflammatory response in patients. Collectively, berberine might be used as an alternative therapeutic strategy for the management of I/R injury.
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Berberina/farmacología , Berberina/uso terapéutico , Sustancias Protectoras/farmacología , Sustancias Protectoras/uso terapéutico , Daño por Reperfusión/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Humanos , Modelos Animales , Transducción de Señal/efectos de los fármacosRESUMEN
Autophagy is an evolutionarily conserved catabolic mechanism, by which eukaryotic cells recycle or degrades internal constituents through membrane-trafficking pathway. Thus, autophagy provides the cells with a sustainable source of biomolecules and energy for the maintenance of homeostasis under stressful conditions such as tumor microenvironment. Recent findings revealed a close relationship between autophagy and malignant transformation. However, due to the complex dual role of autophagy in tumor survival or cell death, efforts to develop efficient treatment strategies targeting the autophagy/cancer relation have largely been unsuccessful. Here we review the two-faced role of autophagy in cancer as a tumor suppressor or as a pro-oncogenic mechanism. In this sense, we also review the shared regulatory pathways that play a role in autophagy and malignant transformation. Finally, anti-cancer therapeutic agents used as either inhibitors or inducers of autophagy have been discussed.
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Autofagia/efectos de los fármacos , Autofagia/fisiología , Neoplasias/metabolismo , Animales , Antineoplásicos , Genes Supresores de Tumor , Humanos , Terapia Molecular Dirigida , Neoplasias/terapia , Oncogenes , Microambiente TumoralRESUMEN
Formation of thrombosis is associated with activation of platelets and endothelial cells. The effect of LongShengZhi Capsule (LSZ), a traditional Chinese medicine used for treatment of vascular diseases, on thrombosis was investigated in this study. BALB/c mice were induced thrombosis by injection of carrageenan while receiving pre or simultaneous LSZ treatment. We also compared the therapeutic effects of LSZ and clopidogrel on formed thrombi. LSZ inhibited carrageenan-induced thrombi in mouse tissue vessels. In addition, LSZ but not clopidogrel reduced formed thrombi with a short time window. The reduction of thrombi by LSZ was associated with reduced serum P-selectin, reduced expression of TNF-α and P-selectin and activated matrix metalloproteinase 2 expression in tissues. In vitro, LSZ decreased thrombin-induced human platelet clot retraction which was associated with inactivation of AKT and ERK1/2. LSZ also reduced adhesion of platelets or THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein or lipopolysaccharide. The anti-adherent actions of LSZ was attributed to reduction of oxidative stress, expression of platelet receptors (P2Y12, PAR4 and CD36) and AKT activity in platelets. LSZ also reduced adhesion molecules or tissue factor but activated tissue factor pathway inhibitor expression in HUVECs. Taken together, our study demonstrates the antithrombotic properties of LSZ by reducing activation of platelets and endothelial cells, and suggests its potential application in clinics.
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Medicamentos Herbarios Chinos/uso terapéutico , Células Endoteliales/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Trombosis/tratamiento farmacológico , Animales , Plaquetas/efectos de los fármacos , Plaquetas/patología , Carragenina , Células Endoteliales/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones Endogámicos BALB C , Trombosis/inducido químicamente , Trombosis/patologíaRESUMEN
Tranilast is an anti-allergy medication that inhibits the release of chemical mediators such as histamine. However, the mechanisms underlying its anti-allergy effects are not fully understood. Interleukin (IL)-33, a novel member of the IL-1 cytokine family, promotes T helper type 2 immune responses and plays a pathogenic role in allergic disorders. In the present study, we examined the effects of tranilast on IL-33 production by RAW264.7 macrophages. Lipopolysaccharide (LPS) increased both IL-33 mRNA expression and IL-33 protein synthesis. Tranilast significantly inhibited LPS-induced IL-33 protein production by RAW264.7 macrophages in a dose-dependent manner; these same effects were observed on IL-33 mRNA levels in RAW264.7 macrophages and a primary culture of macrophages. LPS markedly activated Akt in RAW264.7 macrophages, whereas tranilast suppressed LPS-induced Akt activation. The effects of tranilast on Akt activation appeared to be responsible for the decrease in IL-33 production. Our present findings suggest that the inhibition of IL-33 production by tranilast might contribute to the anti-allergy effects of this medication.
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Interleucina-33/biosíntesis , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , ortoaminobenzoatos/farmacología , Animales , Células de la Médula Ósea/citología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-33/genética , Lipopolisacáridos/farmacología , Macrófagos/citología , Ratones , Células RAW 264.7 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The present study was performed to investigate the potential role of Danshensu in therapeutic angiogenesis in ischemic myocardium and endothelial progenitor cells (EPCs) function. The rat model of myocardial infarction (MI) injury was induced by left anterior descending coronary artery ligation for 14 days. Danshensu significantly alleviated myocardial ischemia injury by ameliorating left ventricular function and reducing infarct size. Furthermore, Danshensu potentiated post-ischemia neovascularization as evidenced by increased microvessel density in infarction boundary zone, as well as the expression of marker proteins vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Moreover, Danshensu notably promoted stromal cell-derived factor-1α (SDF-1α) level in plasma and C-X-C chemokine receptor type 4 (CXCR4) expression in peri-infarction myocardium, and AMD3100 (CXCR4 antagonist) could reverse the angiogenic and cardioprotective effects of Danshensu. For in vitro study, EPCs were isolated from bone marrow of rats. On the one hand, Danshensu provided significant cytoprotection against hypoxia insult by boosting EPCs viability and inhibiting apoptosis, and upregulated Akt phosphorylation. On the other hand, Danshensu enhanced proangiogenic functions of EPCs on cell migration and tube formation, and increased SDF-1α and CXCR4 expression. Likewise, the cytoprotection and proangiogenic functions of Danshensu on EPCs were partly negated by LY294002 (PI3K antagonist) and CXCR4 siRNA, respectively. Taken together, our results suggested that the cardioprotection of Danshensu in MI rats may be related to promoting myocardial neovascularization. The possible mechanisms may involve improving EPCs survival in hypoxia condition through Akt phosphorylation, and accelerating EPCs proangiogenic functions through SDF-1α/CXCR4 axis.
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Quimiocina CXCL12/metabolismo , Células Endoteliales/patología , Lactatos/farmacología , Infarto del Miocardio/fisiopatología , Neovascularización Patológica/tratamiento farmacológico , Receptores CXCR4/metabolismo , Células Madre/patología , Animales , Movimiento Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Corazón/efectos de los fármacos , Corazón/fisiopatología , Lactatos/uso terapéutico , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
AIMS: Endotoxemia and its pro-fibrogenic signaling play a significant role in the development of hepatic fibrosis. This study investigated whether lipopolysaccharide (LPS) directly activate cultured HSC-T6 hepatic stellate cells (HSCs) through triggering Smad-dependent pro-fibrogenic signaling pathway. MAIN METHODS: Direct cell counting and assays for cell proliferation and migration were used to measure the effects of LPS on HSC behaviors. Quantitative PCR, Western blot, and gelatin zymography were used to quantify the molecular effects of LPS on expression of HSC activation markers and signaling activity. KEY FINDINGS: Long-term exposure to LPS exhibited moderately stimulatory effect on HSC cell growth. A wound-healing cell migration assay showed that LPS suppressed HSC-T6 cell migration. qPCR and Western blotting detection indicated that LPS treatment induced upregulation of type I and IV collagens, α-smooth muscle actin (α-SMA), and matrix metalloproteinase-9 (MMP-9). Gelatin zymography confirmed that LPS elevated MMP-9, but not MMP-2 gelatinolytic activity. Moreover, LPS immediately stimulated Akt, EKR1/2, JNK, p38 MAPK, and Smad2 hyperphosphorylation, supporting that LPS directly triggers pro-fibrogenic Smad signaling cascade without TGF-ß1 stimulation. Kinase blockade experiments demonstrated the involvement of PI3K/Akt, JNK, p38 MAPK, but not ERK1/2 signaling activation in the LPS-elicited Smad2 phosphorylation as well as the overexpression of type I collagen and α-SMA in HSC-T6 cells. SIGNIFICANCE: These findings demonstrate that LPS exerts pro-fibrogenic effect through activation and transformation of HSCs. The tissue-remodeling effect of LPS may be attributable to its ability to activate non-canonical Smad pathway through PI3K/Akt and MAPK signaling cascades.
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Proliferación Celular/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Lipopolisacáridos/farmacología , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Animales , Western Blotting , Línea Celular , Movimiento Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Endotoxemia/fisiopatología , Células Estrelladas Hepáticas/metabolismo , Lipopolisacáridos/administración & dosificación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Sensorineural hearing loss (SNHL) is mainly caused by the damage of cochlear hair cells (HCs). As HCs and supporting cells (SCs) do not proliferate in postnatal mammals, the loss of HCs and SCs is irreversible, emphasizing the importance of preserving their numbers to prevent SNHL. It is known that insulin-like growth factor 1 (IGF1) is instrumental in the treatment of SNHL. Our previous study indicates that IGF1 protects HCs against aminoglycoside by activating IGF1 receptor and its two major downstream pathways, PI3K/AKT and MEK/ERK, in SCs, which results in the upregulation of the expression of the Netrin1-encoding gene (Ntn1). However, the mechanisms underlying IGF1-induced protection of HCs via SC activation as well as the role of NTN1 in this process have not been elucidated. Here, we demonstrated that NTN1, similar to IGF1, promoted HC survival. NTN1 blocking antibody attenuated IGF1-induced HC protection from aminoglycoside, indicating that NTN1 is the effector molecule of IGF1 signaling during HC protection. In situ hybridization demonstrated that IGF1 potently induced Ntn1 expression in SCs. NTN1 receptors were abundantly expressed in the cochlea; among them, UNC5B mediated IGF1 protective effects on HCs, as NTN1 binding to UNC5B inhibited HC apoptosis. These results provide new insights into the mechanisms underlying IGF1 protection of cochlear HCs, suggesting a possibility of using NTN1 as a new treatment for SNHL.
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Cóclea/citología , Células Ciliadas Auditivas/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/farmacología , Animales , Animales Recién Nacidos , Antibacterianos/farmacología , Anticuerpos/farmacología , Caspasa 3/metabolismo , Recuento de Células , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Ratones , Ratones Endogámicos ICR , Neomicina/farmacología , Factores de Crecimiento Nervioso/inmunología , Receptores de Netrina , Netrina-1 , Técnicas de Cultivo de Órganos , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
For nanoparticles to be successful in combating diseases in the clinic in the 21st century and beyond, they must localize to target areas of the body and avoid damaging non-target, healthy tissues. Both soft and stiff, bio-degradable and non-biodegradable nanoparticles are anticipated to be used to this end. It has been shown that stiff, non-biodegradable nanoparticles cause reactive oxygen species (ROS) generation and autophagy in a variety of cell lines in vitro. Both responses can lead to significant remodeling of the cytosol and even apoptosis. Thus these are crucial cellular functions to understand. Improved assays have uncovered crucial roles of the Akt/mTOR signaling pathway in both ROS generation and autophagy initiation after cells have internalized stiff, non-biodegradable nanoparticles over varying geometries in culture. Of particular - yet unresolved - interest is how these nanoparticles cause the activation of these pathways. This article reviews the most recent advances in nanoparticle generation of ROS and autophagy initiation with a focus on stiff, non-biodegradable technologies. We provide experimental guidelines to the reader for fleshing out the effects of their nanoparticles on the above pathways with the goal of tuning nanoparticle design.
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Autofagia , Nanopartículas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Humanos , Nanopartículas/químicaRESUMEN
ETHNO-PHARMACOLOGICAL RELEVANCE: Lactucopicrin is one of constitutes in Cichorium intybus L, which is commonly known as chicory in worldwide. It has been used for traditional usage such as antianalgesics, antidepressants and antihyperglycemics AIM OF STUDY: We investigated the neurotrophin-mediated neuroprotective effect of lactucopicrin in in vitro and examined for the underlying mechanism. MATERIALS AND METHOD: To verify the neuroprotective effect of lactucopicrin, we investigated the inhibitory AChE activity, neurite outgrowth-related downstream signaling in murine neuroblastoma N2a and neurotrophins secretion in rat C6 glioma cells. RESULTS: Lactucopicrin inhibited the AChE activity and increased intracellular Ca2+ levels with a substantial rise in muscarinic acetylcholine receptor M1 (CHRM1) expression in N2a cells. Moreover, lactucopicrin actively promoted neurite outgrowth via Ca2+-mediated activation of Ca2+/calmodulin-dependent protein kinase-II (CaMKII). It further activates transcription factor 1 (ATF1) along with modulating the levels of tropomyosin receptor kinase A, extracellular signal-regulated kinase 1 and 2, AKT, and synaptophysin 1 in N2a cells. Additionally, the levels of neurotrophins including NGF, BDNF, and NT3 were increased by treatment of lactucopicrin in C6 cells. The effects of lactucopicrin on NGF secretion and neuritogenesis were maintained even in the presence of phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002, indicating that lactucopicrin exerts its effect on neuritogenesis in a PI3K-independent manner. CONCLUSION: Our results suggest that the natural compound lactucopicrin may be a promising neurotrophin-mediated neuroprotective candidate for neurodegenerative diseases.
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Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Calcio/metabolismo , Inhibidores de la Colinesterasa/farmacología , Lactonas/farmacología , Neuritas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Proteínas/fisiología , Sesquiterpenos/farmacología , Animales , Línea Celular Tumoral , Ratones , Neuritas/fisiología , RatasRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Evodiae Fructus (EF) is the dried, unripe fruit of Evodia rutaecarpa Benth., and one of the main components of traditional herbal prescriptions issued for the treatment of sterility caused by irregular menstruation in Korea. However, scientific evidence regarding the efficacy and action mechanism of EF is lacking. AIM OF THE STUDY: In this study, the authors established an in vitro screening tool to identify promising new drug candidates in herbal medicines for the prevention and treatment of premature ovarian failure. The protective effects of EF extracts against 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity were investigated and the molecular mechanism responsible was sought. MATERIAL AND METHODS: EF extract was prepared by boiling EF in water and its quality was confirmed by high performance liquid chromatography. CHO-K1 (Chinese hamster ovary cells) and COV434 (human ovarian granulosa cells) cells were plated, pretreated with EF extract for 2h and then treated with 1.5mM or 0.5mM VCD for 24h, respectively. Cell viabilities were measured using an MTT assay, and protein levels were determined by western blotting. RESULTS: VCD significantly suppressed the viability of both CHO-K1 and COV434 cells in a dose-dependent manner and induced the apoptosis of CHO-K1 cells at 1.5mM. EF extract dose-dependently blocked the ovotoxicity induced by treatment with VCD. Furthermore, EF extract significantly activated Akt and downstream effectors such as mTOR and GSK-3ß in CHO-K1 cells. The ability of EF extract to prevent cytotoxicity by VCD was antagonized by pretreatment of LY294002, a PI3K/Akt inhibitor. CONCLUSION: EF has the ability to protect ovary cells against VCD-induced ovotoxicity, probably via Akt activation. These results suggest that the beneficial effects of EF might be useful for preventing premature ovarian failure or unexplained infertility caused by environmental factors.
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Ciclohexenos/toxicidad , Activación Enzimática/efectos de los fármacos , Evodia/química , Células de la Granulosa/efectos de los fármacos , Ovario/efectos de los fármacos , Extractos Vegetales/farmacología , Sustancias Protectoras/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Compuestos de Vinilo/toxicidad , Animales , Apoptosis/efectos de los fármacos , Células CHO , Línea Celular , Cricetinae , Cricetulus , Femenino , HumanosRESUMEN
Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2µg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piranos/farmacología , Timidilato Sintasa/metabolismo , Antibacterianos/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Regulación hacia Abajo , Quimioterapia Combinada , Humanos , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Timidilato Sintasa/genéticaRESUMEN
Exogenous hydrogen sulfide (H2S) protects against myocardial ischemia/reperfusion injury but the mechanism of action is unclear. The present study investigated the effect of GYY4137, a slow-releasing H2S donor, on myocardial infarction given specifically at reperfusion and the signalling pathway involved. Thiobutabarbital-anesthetised rats were subjected to 30min of left coronary artery occlusion and 2h reperfusion. Infarct size was assessed by tetrazolium staining. In the first study, animals randomly received either no treatment or GYY4137 (26.6, 133 or 266µmolkg(-1)) by intravenous injection 10min before reperfusion. In a second series, involvement of PI3K and NO signalling were interrogated by concomitant administration of LY294002 or L-NAME respectively and the effects on the phosphorylation of Akt, eNOS, GSK-3ß and ERK1/2 during early reperfusion were assessed by immunoblotting. GYY4137 266µmolkg(-1) significantly limited infarct size by 47% compared to control hearts (P<0.01). In GYY4137-treated hearts, phosphorylation of Akt, eNOS and GSK-3ß was increased 2.8, 2.2 and 2.2 fold respectively at early reperfusion. Co-administration of L-NAME and GYY4137 attenuated the cardioprotection afforded by GYY4137, associated with attenuated phosphorylation of eNOS. LY294002 totally abrogated the infarct-limiting effect of GYY4137 and inhibited Akt, eNOS and GSK-3ß phosphorylation. These data are the first to demonstrate that GYY4137 protects the heart against lethal reperfusion injury through activation of PI3K/Akt signalling, with partial dependency on NO signalling and inhibition of GSK-3ß during early reperfusion. H2S-based therapeutic approaches may have value as adjuncts to reperfusion in the treatment of acute myocardial infarction.
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Sulfuro de Hidrógeno/metabolismo , Morfolinas/farmacología , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Compuestos Organotiofosforados/farmacología , Sustancias Protectoras/farmacología , Animales , Citoprotección , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Hemodinámica/efectos de los fármacos , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
This study focused on the anti-proliferation effects of ursolic acid (UA) in rat primary vascular smooth muscle cells (VSMCs) and investigated underlying molecular mechanism of action. Rat primary VSMCs were pretreated with UA (10, 20 or 30µM) or amino guanidine (AG, 50µM) for 12h or with PI3K inhibitor LY294002 for 30min or with Akt inhibitor MK2206 for 24h, then 10% fetal bovine serum was used to induce proliferation. CCK-8 was used to assess cell proliferation. To explore the mechanism, cells were treated with UA (10, 20 or 30µM), LY294002 or MK2206, or transient transfected to inhibit miRNA-21 (miRNA-21) or to overexpress PTEN, then quantitative real-time PCR was used to assess the mRNA levels of miRNA-21 and phosphatase and tensin homolog (PTEN) for cells treated with UA or miRNA-21 inhibitor; western blotting was used to measure the protein levels of PTEN and PI3K. UA exerted significant anti-proliferation effects in rat primary VSMCs. Furthermore, UA inhibited the expression of miRNA-21 and subsequently enhanced the expression of PTEN. PTEN was found to inhibit the expression of PI3K. In conclusion, UA exerts anti-proliferation effects in rat primary VSMCs, which is associated with the inhibition of miRNA-21 expression and modulation of PTEN/PI3K signaling pathway.