RESUMEN
Platelet hyper-reactivity and neutrophil extracellular trap (NET) formation contribute to the development of thromboembolic diseases for patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study investigated the pathophysiological effects of SARS-CoV-2 surface protein components and the viral double-stranded RNA (dsRNA) on platelet aggregation and NET formation. Traditional Chinese medicine (TCM) with anti-viral effects was also delineated. The treatment of human washed platelets with SARS-CoV-2 spike protein S1 or the ectodomain S1 + S2 regions neither caused platelet aggregation nor enhanced agonists-stimulated platelet aggregation. Moreover, NET formation can be induced by polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analog of viral dsRNA, but not by the pseudovirus composed of SARS-CoV-2 spike, envelope, and membrane proteins. To search for TCM with anti-NET activity, the plant Melastoma malabathricum L. which has anticoagulant activity was partially purified by fractionation. One of the fractions inhibited poly(I:C)-induced NET formation in a dose-dependent manner. This study implicates that SARS-CoV-2 structural proteins alone are not sufficient to promote NET and platelet activation. Instead, dsRNA formed during viral replication stimulates NET formation. This study also sheds new insight into using the active components of Melastoma malabathricum L. with anti-NET activity in the battle of thromboembolic diseases associated with SARS-CoV-2 infection.
RESUMEN
The over-activated neutrophils through G-protein-coupled receptors (GPCRs) caused inflammation or tissue damage. Therefore, GPCRs or their downstream molecules are major targets for inhibiting uncontrolled neutrophil activation. Our studies investigate the action and underlying mechanism of larixol, a diterpene extract from the root of euphorbia formosana, on fMLP-induced neutrophil respiratory burst, chemotaxis, and granular release. The immunoprecipitation assay was performed to investigate whether larixol inhibits fMLP-induced respiratory burst by interfering with the interaction of fMLP receptor Gi-protein ßγ subunits with its downstream molecules. Briefly, larixol inhibited fMLP (0.1 µM)-induced superoxide anion production (IC50:1.98 ± 0.14 µM), the release of cathepsin G (IC50:2.76 ± 0.15 µM) and chemotaxis in a concentration-dependent manner; however, larixol did not inhibit these functions induced by PMA (100 nM). Larixol inhibited fMLP-induced Src kinase phosphorylation. Therefore, larixol attenuated the downstream signaling of Src kinases, ERK1/2, p38, and AKT phosphorylation. Moreover, larixol inhibited fMLP-induced intracellular calcium mobilization, PKC phosphorylation, and p47phox translocation from the cytosol to the plasma membrane. Larixol inhibited the interaction of the ßγ subunits of Gi-protein of fMLP receptor with Src kinase or with PLCß by the immunoprecipitation and duolink assay. Furthermore, larixol did not antagonize the formyl peptide receptors. Larixol did not increase cyclic nucleotide levels in neutrophils. These results suggest that larixol modulated fMLP-induced neutrophils superoxide anion production, chemotaxis, and granular releases by interrupting the interaction of the ßγ subunits of Gi-protein with downstream signaling of the fMLP receptor.
Asunto(s)
Diterpenos , Receptores de Formil Péptido , Quimiotaxis , Humanos , N-Formilmetionina Leucil-Fenilalanina/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Fosforilación , Receptores de Formil Péptido/metabolismo , Superóxidos/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Three new and uncommon chromone analogs, epiremisporine F (1), epiremisporine G (2), and epiremisporine H (3), were isolated from marine-origin Penicillium citrinum. Among the isolated compounds, compounds 2-3 remarkably suppressed fMLP-induced superoxide anion generation by human neutrophils, with IC50 values of 31.68 ± 2.53, and 33.52 ± 0.42 µM, respectively. Compound 3 exhibited cytotoxic activities against human colon carcinoma (HT-29) and non-small lung cancer cell (A549) with IC50 values of 21.17 ± 4.89 and 31.43 ± 3.01 µM, respectively, and Western blot assay confirmed that compound 3 obviously induced apoptosis of HT-29 cells, via Bcl-2, Bax, and caspase 3 signaling cascades.
Asunto(s)
Antiinflamatorios/farmacología , Antineoplásicos/farmacología , Organismos Acuáticos/química , Cromonas/farmacología , Penicillium/química , Línea Celular Tumoral/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Neutrófilos/efectos de los fármacosRESUMEN
The uncontrol respiratory burst in neutrophils can lead to inflammation and tissue damage. This study investigates the effect and the underlying mechanism of ε-viniferin, a lignan from the root of Vitis thunbergii var. thunbergii, inhibits N-formyl-L-methionyl-L-leucyl-l-phenylalanine (fMLP) induced respiratory burst by antagonizing formyl peptide receptor 1 in human neutrophils. Briefly, ε-viniferin specifically inhibited fMLP (0.1 µM: formyl peptide receptor 1 agonist or 1 µM: formyl peptide receptor 1, 2 agonist)-induced superoxide anion production in a concentration-dependent manner (IC50 = 2.30 ± 0.96 or 9.80 ± 0.21 µM, respectively) without affecting this induced by formyl peptide receptor 2 agonist (WKYMVM). ε-viniferin inhibited fMLP (0.1 µM)-induced phosphorylation of ERK, Akt, Src or intracellular calcium mobilization without affecting these caused by WKYMVM. The synergistic suppression of fMLP (1 µM)-induced superoxide anion production was observed only in the combination of ε-viniferin and formyl peptide receptor 2 antagonist (WRW4) but not in combination of ε-viniferin and formyl peptide receptor 1 antagonist (cyclosporine H). ε-viniferin inhibited FITC-fMLP binding to formyl peptide receptors. Moreover, the synergistic suppression of FITC-fMLP binding was observation only in the combination of ε-viniferin and WRW4 but not in other combinations. ATPγS induced superoxide anion production through formyl peptide receptor 1 in fMLP desensitized neutrophils and this effect was inhibited by ε-viniferin. The concentration-response curve of fMLP-induced superoxide anion was not parallel shifted by ε-viniferin. Furthermore, the inhibiting effect of ε-viniferin on fMLP-induced superoxide anion production was reversible. These results suggest that ε-viniferin is an antagonist of formyl peptide receptor 1 in a reversible and non-competitive manner.
Asunto(s)
Antiinflamatorios/farmacología , Benzofuranos/farmacología , Terapia Molecular Dirigida , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Receptores de Formil Péptido/antagonistas & inhibidores , Estilbenos/farmacología , Secuencia de Aminoácidos , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Oligopéptidos/química , Oligopéptidos/farmacología , Superóxidos/metabolismoRESUMEN
BACKGROUND: Bidirectional integrin αIIbß3 signaling is essential for platelet activation. The platelet adaptor protein Disabled-2 (Dab2) is a key regulator of integrin signaling and is phosphorylated at serine 24 in eukaryotic cells. However, the mechanistic insight and function of Dab2-serine 24 phosphorylation (Dab2-pSer24) in platelet biology are barely understood. This study aimed to define whether and how Dab2 is phosphorylated at Ser24 during platelet activation and to investigate the effect of Dab2-pSer24 on platelet function. RESULTS: An antibody with confirmed specificity for Dab2-pSer24 was generated. By using this antibody as a tool, we showed that protein kinase C (PKC)-mediated Dab2-pSer24 was a conservative signaling event when human platelets were activated by the platelet agonists such as thrombin, collagen, ADP, 12-O-tetradecanoylphorbol-13-acetate, and the thromboxane A2 activator U46619. The agonists-stimulated Dab2-pSer24 was attenuated by pretreatment of platelets with the RGDS peptide which inhibits integrin outside-in signaling by competitive binding of integrin αIIb with fibrinogen. Direct activation of platelet integrin outside-in signaling by combined treatment of platelets with manganese dichloride and fibrinogen or by spreading of platelets on fibrinogen also resulted in Dab2-pSer24. These findings implicate that Dab2-pSer24 was associated with the outside-in signaling of integrin. Further analysis revealed that Dab2-pSer24 was downstream of Src-PKC-axis and phospholipase D1 underlying the integrin αIIbß3 outside-in signaling. A membrane penetrating peptide R11-Ser24 which contained 11 repeats of arginine linked to the Dab2-Ser24 phosphorylation site and its flanking sequences (RRRRRRRRRRR19APKAPSKKEKK29) and the R11-S24A peptide with Ser24Ala mutation were designed to elucidate the functions of Dab2-pSer24. R11-Ser24 but not R11-S24A inhibited agonists-stimulated Dab2-pSer24 and consequently suppressed platelet spreading on fibrinogen, with no effect on platelet aggregation and fibrinogen binding. Notably, Ser24 and the previously reported Ser723 phosphorylation (Dab2-pSer723) occurred exclusively in a single Dab2 molecule and resulted in distinctive subcellular distribution and function of Dab2. Dab2-pSer723 was mainly distributed in the cytosol of activated platelets and associated with integrin inside-out signaling, while Dab2-pSer24 was mainly distributed in the membrane fraction of activated platelets and associated with integrin outside-in signaling. CONCLUSIONS: These findings demonstrate for the first time that Dab2-pSer24 is conservative in integrin αIIbß3 outside-in signaling during platelet activation and plays a novel role in the control of cytoskeleton reorganization and platelet spreading on fibrinogen.
RESUMEN
Three new and rare chromone derivatives, epiremisporine C (1), epiremisporine D (2), and epiremisporine E (3), were isolated from marine-derived Penicillium citrinum, together with four known compounds, epiremisporine B (4), penicitrinone A (5), 8-hydroxy-1-methoxycarbonyl-6-methylxanthone (6), and isoconiochaetone C (7). Among the isolated compounds, compounds 2-5 significantly decreased fMLP-induced superoxide anion generation by human neutrophils, with IC50 values of 6.39 ± 0.40, 8.28 ± 0.29, 3.62 ± 0.61, and 2.67 ± 0.10 µM, respectively. Compounds 3 and 4 exhibited cytotoxic activities with IC50 values of 43.82 ± 6.33 and 32.29 ± 4.83 µM, respectively, against non-small lung cancer cell (A549), and Western blot assay confirmed that compounds 3 and 4 markedly induced apoptosis of A549 cells, through Bcl-2, Bax, and caspase 3 signaling cascades.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Cromonas/química , Cromonas/farmacología , Penicillium/química , Células A549 , Adulto , Antibacterianos/farmacología , Caspasa 3/efectos de los fármacos , Línea Celular Tumoral , Fermentación , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Superóxidos/química , Adulto Joven , Proteína X Asociada a bcl-2/efectos de los fármacosRESUMEN
Tumor cell-induced platelet aggregation (TCIPA) is a mechanism that involves the protection of tumor cells in the circulation and the promotion of tumor cell invasion and metastases. The C-type lectin-like receptor 2 (CLEC-2) that binds podoplanin (PDPN) is on the platelet surface and facilitates the TCIPA. Selective blockage of the PDPN-mediated platelet-tumor cell interaction is thereby a plausible strategy for inhibiting metastases. In a search for antagonists of PDPN- and tumor cell-induced platelet aggregation, traditional Chinese medicines were screened and it was found that the water extract of Artemisia argyi leaves selectively inhibited the PDPN-induced platelet aggregation. Bioactivity-guided fractionation analysis was performed for defining a polysaccharide-containing fraction (AAWAP) characterized by inhibition of PDPN activity and tumor cell-induced platelet aggregation. The pharmacological effects of AAWAP on PDPN-activated CLEC-2 signaling were determined by using Western blot and alpha screening analyses. AAWAP was non-toxic to the cells and platelets and it suppressed PDPN- and tumor cell-induced platelet aggregation by irreversibly blocking the interaction between PDPN and CLEC-2 in a dose-dependent manner. These findings indicate that AAWAP is an antagonist of the PDPN-CLEC-2 interaction. This action by AAWAP may result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.
Asunto(s)
Artemisia/clasificación , Lectinas Tipo C/antagonistas & inhibidores , Glicoproteínas de Membrana/antagonistas & inhibidores , Agregación Plaquetaria/efectos de los fármacos , Polisacáridos/farmacología , Línea Celular Tumoral , Humanos , Fitoquímicos/farmacología , Hojas de la Planta/químicaRESUMEN
Four new 2-(2-phenylethyl)-4H-chromen-4-one derivatives, 6-hydroxy-5-methoxy-2-[2-(4'-methoxyphenyl)ethyl]chromone (1: ), 6,7-dimethoxy-2-[2-(2'-hydroxyphenyl)ethyl]chromone (2: ), 5-hydroxy-6-methoxy-2-[2-(3'-methoxyphenyl)ethyl]-chromone (3: ), and 7-chloro-8-hydroxy-2-[2-(4'-methoxyphenyl)ethyl]chromone (4: ), have been isolated from the resinous wood of Aquilaria sinensis, together with 16 known compounds (5: -20: ). Among these, 7-methoxy-2-[2-(4'-methoxyphenyl)ethyl]chromone (5: ) was isolated from a natural source for the first time. The structures of the new compounds were established by spectroscopic analyses (1D and 2D NMR, HR-ESI-MS, IR, UV). Nine compounds, including 1: showed more than 80% inhibition of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine at 50 µM.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Cromonas/química , Cromonas/farmacología , Resinas de Plantas/química , Thymelaeaceae/química , Adulto , Evaluación Preclínica de Medicamentos/métodos , Humanos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiologíaRESUMEN
This study investigates the effect and the underlying mechanism of 2',3-dihydroxy-5-methoxybiphenyl (RIR-2), a lignan extracted from the roots of Rhaphiolepis indica (L.) Lindl. ex Ker var. tashiroi Hayata ex Matsum. & Hayata (Rosaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst and cathepsin G in human neutrophils. Signaling pathways regulated by RIR-2 which modulated fMLP-induced respiratory burst were evaluated by an interaction between ß subunit of G-protein (Gß) with downstream signaling induced by fMLP and by immunoblotting analysis of the downstream targets of Gß-protein. RIR-2 inhibited fMLP-induced superoxide anion production (IC50:2.57⯱â¯0.22⯵M), cathepsin G release (IC50:18.72⯱â¯3.76⯵M) and migration in a concentration dependent manner. RIR-2 specifically suppresses fMLP-induced Src family kinases phosphorylation by inhibiting the interaction between Gß-protein with Src kinases without inhibiting Src kinases activities, therefore, RIR-2 attenuated the downstream targets of Src kinase, such as phosphorylation of Raf/ERK, AKT, P38, PLCγ2, PKC and translocation Tec, p47ph°x and P40ph°x from the cytosol to the inner leaflet of the plasma membrane. Furthermore, RIR-2 attenuated fMLP-induced intracellular calcium mobilization by inhibiting the interaction between Gß-protein with PLCß2. RIR-2 was not a competitive or allosteric antagonist of fMLP. On the contrary, phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of Src, AKT, P38, PKC and membrane localization of p47ph°x and P40ph°x remained unaffected. RIR-2 specifically modulates fMLP-mediated neutrophil superoxide anion production and cathepsin G release by inhibiting the interaction between Gß-protein with downstream signaling which subsequently interferes with the activation of intracellular calcium, PLCγ2, AKT, p38, PKC, ERK, p47ph°x and p40phox.
Asunto(s)
Catepsina G/metabolismo , Lignanos/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Superóxidos/metabolismo , Calcio/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inhibidores , Neutrófilos/citología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Estallido Respiratorio/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) are high-mortality and life-threatening diseases that are associated with neutrophil activation and accumulation within lung tissue. Emerging evidence indicates that neutrophil-platelet aggregates (NPAs) at sites of injury increase acute inflammation and contribute to the development of ALI. Although numerous studies have increased our understanding of the pathophysiology of ALI, there is still a lack of innovative and useful treatments that reduce mortality, emphasizing that there is an urgent need for novel treatment strategies. In this study, a new series of small compounds of ß-nitrostyrene derivatives (BNSDs) were synthesized, and their anti-inflammatory bioactivities on neutrophils and platelets were evaluated. The new small compound C7 modulates neutrophil function by inhibiting superoxide generation and elastase release. Compound C7 elicits protective effects on LPS-induced paw edema and acute lung injury via the inhibition of neutrophil accumulation, proinflammatory mediator release, platelet aggregation, myeloperoxidase activity, and neutrophil extracellular trap (NET) release. NET formation was identified as the bridge for the critical interactions between neutrophils and platelets by confocal microscopy and flow cytometry. This research provides new insights for elucidating the complicated regulation of neutrophils and platelets in ALI and sheds further light on future drug development strategies for ALI/ARDS and acute inflammatory diseases.
Asunto(s)
Lesión Pulmonar Aguda/tratamiento farmacológico , Plaquetas/efectos de los fármacos , Trampas Extracelulares/metabolismo , Lipopolisacáridos/toxicidad , Neutrófilos/efectos de los fármacos , Edema Pulmonar/tratamiento farmacológico , Estirenos/farmacología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/patología , Animales , Plaquetas/inmunología , Plaquetas/metabolismo , Plaquetas/patología , Adhesión Celular , Células Cultivadas , Trampas Extracelulares/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Edema Pulmonar/inducido químicamente , Edema Pulmonar/inmunología , Edema Pulmonar/patologíaRESUMEN
Two new naphthofuranone derivatives, 11-hydroxy-2-O-methylhibiscolactone A (1) and O-methylhibiscone D (2), have been isolated from the stems of Pachira aquatica, together with 18 known compounds (3-20). The structures of two new compounds were determined through spectroscopic and MS analyses. Among the isolated compounds, 11-hydroxy-2-O-methylhibiscolactone A (1), isohemigossylic acid lactone-7-methyl ether (4), gmelofuran (6), and 5-hydroxyauranetin (8) exhibited inhibition (IC50≤28.84µM) of superoxide anion generation by human neutrophils in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP).
Asunto(s)
Antiinflamatorios/química , Bombacaceae/química , Furanos/química , Neutrófilos/efectos de los fármacos , Superóxidos/metabolismo , Adulto , Antiinflamatorios/aislamiento & purificación , Células Cultivadas , Furanos/aislamiento & purificación , Humanos , Estructura Molecular , N-Formilmetionina Leucil-Fenilalanina , Neutrófilos/metabolismo , Extractos Vegetales/química , Tallos de la Planta/química , Adulto JovenRESUMEN
Two new sesquiterpenoids-13-hydroxycurzerenone (1) and 1-oxocurzerenone (2)-have been isolated from the rhizomes of Curcuma zedoaria, together with 13 known compounds (3-15). The structures of two new compounds were determined through spectroscopic and MS analyses. Among the isolated compounds, 13-hydroxycurzerenone (1), 1-oxocurzerenone (2), curzerenone (3), germacrone (4), curcolone (5), procurcumenol (6), ermanin (7), curcumin (8), and a mixture of stigmast-4-en-3,6-dione (12) and stigmasta-4,22-dien-3,6-dione (13) exhibited inhibition (with inhibition % in the range of 21.28%-67.58%) against collagen-induced platelet aggregation at 100 µM. Compounds 1, 5, 7, 8, and the mixture of 12 and 13 inhibited arachidonic acid (AA)-induced platelet aggregation at 100 µM with inhibition % in the range of 23.44%-95.36%.
Asunto(s)
Curcuma/química , Agregación Plaquetaria/efectos de los fármacos , Rizoma/química , Sesquiterpenos/farmacología , Ácido Araquidónico/efectos adversos , Estructura Molecular , Extractos Vegetales/química , Extractos Vegetales/farmacología , Sesquiterpenos/químicaRESUMEN
Podoplanin (PDPN) enhances tumor metastases by eliciting tumor cell-induced platelet aggregation (TCIPA) through activation of platelet C-type lectin-like receptor 2 (CLEC-2). A novel and non-cytotoxic 5-nitrobenzoate compound 2CP was synthesized that specifically inhibited the PDPN/CLEC-2 interaction and TCIPA with no effect on platelet aggregation stimulated by other platelet agonists. 2CP possessed anti-cancer metastatic activity in vivo and augmented the therapeutic efficacy of cisplatin in the experimental animal model without causing a bleeding risk. Analysis of the molecular action of 2CP further revealed that Akt1/PDK1 and PKCµ were two alternative CLEC-2 signaling pathways mediating PDPN-induced platelet activation. 2CP directly bound to CLEC-2 and, by competing with the same binding pocket of PDPN in CLEC-2, inhibited PDPN-mediated platelet activation. This study provides evidence that 2CP is the first defined platelet antagonist with CLEC-2 binding activity. The augmentation in the therapeutic efficacy of cisplatin by 2CP suggests that a combination of a chemotherapeutic agent and a drug with anti-TCIPA activity such as 2CP may prove clinically effective.
Asunto(s)
Plaquetas/efectos de los fármacos , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Nitrobenzoatos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Plaquetas/metabolismo , Western Blotting , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Invasividad Neoplásica/patología , Agregación Plaquetaria/fisiología , Resonancia por Plasmón de Superficie , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A new limonoid, swietemacrophin (1), was isolated from the seeds of Swietenia macrophylla, together with five known compounds 2-6. The structure of 1 was determined through extensive 1D/2D-NMR and mass-spectrometric analyses. Swietemacrophin (1), humilinolide F (2), 3,6-O,O-diacetylswietenolide (3), 3-O-tigloylswietenolide (4), and swietemahonin E (5) exhibited inhibition (IC50 values≤45.44 µM) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Compounds 1, 4, 5, and swietenine (6) showed potent inhibition with IC50 values≤36.32 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation.
Asunto(s)
Antiinflamatorios/aislamiento & purificación , Limoninas/aislamiento & purificación , Meliaceae/química , Semillas/química , Triterpenos/aislamiento & purificación , Antiinflamatorios/química , Antiinflamatorios/farmacología , Humanos , Concentración 50 Inhibidora , Limoninas/química , Limoninas/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inhibidores , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Extractos Vegetales/química , Cultivo Primario de Células , Relación Estructura-Actividad , Superóxidos/antagonistas & inhibidores , Triterpenos/química , Triterpenos/farmacologíaRESUMEN
The undesirable respiratory burst in neutrophils can lead to inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol (honokiol), a lignan extracted from the stem bark of Magnolia officinalis Rehd. et Wils (Magnoliaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst in human neutrophils. Signaling pathways regulated by honokiol which modulate fMLP-induced respiratory burst and cathepsin G release were evaluated by phosphorylation of Src family kinase induced by fMLP, Src family kinases activities and by immunoblotting analysis of the downstream targets of Src kinase. Briefly, honokiol inhibited fMLP-induced superoxide anion production (IC50 = 9.80 ± 0.21 µM, n = 4), cathepsin G release (IC50 = 14.23 ± 1.43 µM, n = 4) and migration (IC50 = 5.69 ± 1.51 µM, n = 4) in a concentration dependent manner. Further, honokiol specifically suppresses fMLP-induced Lyn (a member of the Src kinase family) phosphorylation, by inhibiting Lyn kinase activity. Consequently, honokiol attenuated the downstream targets of Lyn kinase, such as Tec translocation from the cytosol to the inner leaflet of the plasma membrane, phosphorylation of AKT, P38, PLCγ2, protein kinase C and membrane localization of p47(phox). On the other hand, fMLP-induced phosphorylation of Hck, Fgr kinase activity (other members of Src kinase), downstream phosphorylation of Vav1 and extracellular signal-regulated kinase remained unaffected. In addition, honokiol neither inhibited NADPH oxidase activity nor increased cyclic AMP levels. Honokiol is not a competitive or allosteric antagonist of fMLP. In conclusion, honokiol specifically modulates fMLP-mediated neutrophil activation by inhibiting Lyn activation which subsequently interferes with the activation of PLCγ2, AKT, p38, protein kinase C, and p47(phox).
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Compuestos de Bifenilo/farmacología , Lignanos/farmacología , Neutrófilos/efectos de los fármacos , Familia-src Quinasas/antagonistas & inhibidores , Adulto , Calcio/metabolismo , Catepsina G/metabolismo , Movimiento Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacología , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Fosfolipasa C gamma/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Formil Péptido/metabolismo , Estallido Respiratorio/efectos de los fármacos , Superóxidos/metabolismo , Adulto Joven , Familia-src Quinasas/metabolismoRESUMEN
Muntingia calabura (Tiliaceae) is commercially used in healthcare for the improvement of hypertension, myocardial infarction, spasm, and inflammatory conditions. Its fruits can be processed into jam and the leaves can be used for making tea. In the work reported herein a new biflavan, (M),(2S),(2''S)-,(P),(2S),(2''S)-7,8,3',4',5',7'',8'',3''',4''',5'''-decamethoxy-5,5'' biflavan (1), a new flavone, 4'-hydroxy-7,8,3',5'-tetramethoxyflavone (2), and a new dihydrochalcone, (R)-2',ß-dihydroxy-3',4'-dimethoxydihydrochalcone (3), have been isolated from the stem wood of M. calabura, together with 12 known compounds (4-15). The structures of these new compounds were elucidated by the interpretations of extensive spectroscopic data. Among the isolated compounds, 5-hydroxy-7-methoxyflavone (5), quercetin (6), and (2S)-7-hydroxyflavanone (10) exhibited potent inhibition of fMLP-induced superoxide anion generation by human neutrophils, with IC50 values of 1.77 ± 0.70, 3.82 ± 0.46, and 4.92 ± 1.71 µM, respectively.
RESUMEN
BACKGROUND: Obstructive sleep apnea (OSA) is known to be a risk factor of coronary artery disease. The chemotaxis and adhesion of monocytes to the endothelium in the early atherosclerosis is important. This study aimed to investigate the effect of intermittent hypoxia, the hallmark of OSA, on the chemotaxis and adhesion of monocytes. METHODS: Peripheral blood was sampled from 54 adults enrolled for suspected OSA. RNA was prepared from the isolated monocytes for the analysis of C-C chemokine receptor 2 (CCR2). The effect of intermittent hypoxia on the regulation and function of CCR2 was investigated on THP-1 monocytic cells and monocytes. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. Transwell filter migration assay and cell adhesion assay were performed to study the chemotaxis and adhesion of monocytes. RESULTS: Monocytic CCR2 gene expression was found to be increased in severe OSA patients and higher levels were detected after sleep. Intermittent hypoxia increased the CCR2 expression in THP-1 monocytic cells even in the presence of TNF-α and CRP. Intermittent hypoxia also promoted the MCP-1-mediated chemotaxis and adhesion of monocytes to endothelial cells. Furthermore, inhibitor for p42/44 MAPK or p38 MAPK suppressed the activation of monocytic CCR2 expression by intermittent hypoxia. CONCLUSIONS: This is the first study to demonstrate the increase of CCR2 gene expression in monocytes of severe OSA patients. Monocytic CCR2 gene expression can be induced under intermittent hypoxia which contributes to the chemotaxis and adhesion of monocytes.
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Monocitos/fisiología , Receptores CCR2/genética , Receptores CCR2/metabolismo , Apnea Obstructiva del Sueño/sangre , Adulto , Adhesión Celular , Hipoxia de la Célula , Células Cultivadas , Quimiotaxis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Apnea Obstructiva del Sueño/genética , Apnea Obstructiva del Sueño/metabolismo , Regulación hacia ArribaRESUMEN
Two new pterosin sesquiterpenes, (2S)-13-hydroxypterosin A (1) and (2S,3S)-12-hydroxypterosin Q (2), were isolated from the whole plants of Pteris ensiformis, together with six known compounds. The structures of 1 and 2 were determined through extensive 1D/2D-NMR and MS analyses. Compound 2 exhibited antitubercular activity (MIC 6.25â µg/ml) against Mycobacterium tuberculosis H37 Rv in vitro.
Asunto(s)
Antituberculosos , Indanos/química , Mycobacterium tuberculosis/efectos de los fármacos , Extractos Vegetales , Pteris/química , Sesquiterpenos/química , Sesquiterpenos/farmacología , Antituberculosos/química , Antituberculosos/aislamiento & purificación , Antituberculosos/farmacología , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Extractos Vegetales/química , Extractos Vegetales/farmacología , Pteris/metabolismo , Sesquiterpenos/aislamiento & purificaciónRESUMEN
Two new triterpenoids, 2α,3ß-dihydroxyolean-11,13(18)-dien-19ß,28-olide (1) and 3ß,5ß-dihydroxyglutinol (2), together with eight known compounds (3-10) were isolated from the roots of Rhaphiolepis indica var. tashiroi (Rosaceae). The structures of 1-10 were determined by spectroscopic techniques. Among these isolates, 2α,3ß-dihydroxyolean-13(18)-en-28-oic acid (9) exhibited inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide production, with an IC50 value of 16.50 µM.
Asunto(s)
Antiinflamatorios/farmacología , Raíces de Plantas/química , Rosaceae/química , Triterpenos/farmacología , Adulto , Antiinflamatorios/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Humanos , Concentración 50 Inhibidora , Espectroscopía de Protones por Resonancia Magnética , Superóxidos/metabolismo , Triterpenos/química , Adulto JovenRESUMEN
Ondansetron is a 5-HT3 receptor antagonist with potent antiemetic, analgesic, and antiphlogistic effects. Literature concerning 5-HT3 antagonists on platelets is limited. In this report we examined the pharmacological effects of ondansetron on human washed platelets. Platelet aggregation induced by thrombin (0.1 U/mL), collagen (2 µg/mL), arachidonic acid (0.5mM), ADP (10 µM), or U46619 (2 µM) was observed. The effects of ondansetron on platelet aggregation and ATP release were investigated at different concentrations. Cytosolic Ca(2+) influx concentration, TXB2, IP3, and the levels of cAMP and cGMP were monitored, and flow cytometric analysis and immunoblotting were performed to investigate downstream signaling components. Our results showed that ondansetron, in a concentration-dependent manner, inhibited agonist-induced platelet aggregation. At 75 µM, ondansetron significantly attenuated intracellular Ca(2+) mobilization, thromboxane B2 formation, and ATP release by human washed platelets activated by thrombin, collagen, or U46619, whereas it only partially attenuated arachidonic acid-driven platelet activation. Administration of ondansetron resulted in attenuated IP3 production in the washed platelets stimulated by thrombin, as determined by reduced IP1 levels, as well as diminished p38 and ERK2 phosphorylation in response to thrombin. No effect of ondansetron on the levels of either cAMP or cGMP in washed platelets was observed. Furthermore, ondansetron-mediated inhibition of platelet aggregation was not impacted by SR 57227A, the 5-HT3 agonist. Thus, rather than involving the 5-HT3-dependent pathway, the negative effect of ondansetron on platelet aggregation is instead manifested through the attenuation of agonist-induced IP3 production and MAPK (p38 and ERK2) phosphorylation that results in suppressed intracellular Ca(2+) mobilization, TXB2 formation, and ATP release.