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1.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064436

RESUMO

2-Arachidonyl-lysophosphatidylethanolamine, shortly 2-ARA-LPE, is a polyunsaturated lysophosphatidylethanolamine. 2-ARA-LPE has a very long chain arachidonic acid, formed by an ester bond at the sn-2 position. It has been reported that 2-ARA-LPE has anti-inflammatory effects in a zymosan-induced peritonitis model. However, it's action mechanisms are poorly investigated. Recently, resolution of inflammation is considered to be an active process driven by M2 polarized macrophages. Therefore, we have investigated whether 2-ARA-LPE acts on macrophages for anti-inflammation, whether 2-ARA-LPE modulates macrophage phenotypes to reduce inflammation, and whether 2-ARA-LPE is anti-inflammatory in a carrageenan-induced paw edema model. In mouse peritoneal macrophages, 2-ARA-LPE was found to inhibit lipopolysaccharide (LPS)-induced M1 macrophage polarization, but not induce M2 polarization. 2-ARA-LPE inhibited the inductions of inducible nitric oxide synthase and cyclooxygenase-2 in mouse peritoneal macrophages at the mRNA and protein levels. Furthermore, products of the two genes, nitric oxide and prostaglandin E2, were also inhibited by 2-ARA-LPE. However, 1-oleoyl-LPE did not show any activity on the macrophage polarization and inflammatory responses. The anti-inflammatory activity of 2-ARA-LPE was also verified in vivo in a carrageenan-induced paw edema model. 2-ARA-LPE inhibits LPS-induced M1 polarization, which contributes to anti-inflammation and suppresses the carrageenan-induced paw edema in vivo.


Assuntos
Anti-Inflamatórios/farmacologia , Ácidos Araquidônicos/farmacologia , Edema/tratamento farmacológico , Lisofosfolipídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Ácidos Araquidônicos/química , Carragenina/administração & dosagem , Ciclo-Oxigenase 2/imunologia , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Edema/induzido quimicamente , Edema/imunologia , Edema/patologia , Membro Posterior/efeitos dos fármacos , Membro Posterior/imunologia , Membro Posterior/metabolismo , Subunidade p35 da Interleucina-12/antagonistas & inibidores , Subunidade p35 da Interleucina-12/imunologia , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Lisofosfolipídeos/química , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/imunologia , Cultura Primária de Células , Resultado do Tratamento
2.
Molecules ; 26(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067155

RESUMO

The aim of this study was to evaluate the involvement of nanoparticles prepared from Allium cepa L. as anti-inflammatory agents. In the present study, we identified nanoparticles from Allium cepa L. using the ultracentrifugation exosome purification method. The nanoparticles were referred to as 17,000× g and 200,000× g precipitates, and they contained quercetins, proteins, lipids, and small-sized RNA. The nanoparticles inhibited nitric oxide production from lipopolysaccharide (LPS)-stimulated RAW264 cells without cytotoxic properties. Cellular incorporation was confirmed by laser microscopic observation after PKH26 staining. The inhibition of caveolae-dependent endocytosis and macropinocytosis significantly prevented the incorporation of the nanoparticles but had no effect on the inhibition of nitric oxide in RAW264 cells. Collectively, the identified nanoparticles were capable of inhibiting the LPS response via extracellular mechanisms. Taken together, the way of consuming Allium cepa L. without collapsing the nanoparticles is expected to provide an efficient anti-inflammatory effect.


Assuntos
Endocitose , Espaço Intracelular/metabolismo , Nanopartículas/química , Nitratos/metabolismo , Cebolas/química , Animais , Clatrina/metabolismo , Lipopolissacarídeos , Camundongos , Óxido Nítrico/biossíntese , Quercetina/análise , Células RAW 264.7
3.
Molecules ; 26(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925453

RESUMO

Two light-activated NO donors [RuCl(qn)(Lbpy)(NO)]X with 8-hydroxyquinoline (qn) and 2,2'-bipyridine derivatives (Lbpy) as co-ligands were synthesized (Lbpy1 = 4,4'-dicarboxyl-2,2'-dipyridine, X = Cl- and Lbpy2 = 4,4'-dimethoxycarbonyl-2,2'-dipyridine, X = NO3-), and characterized using ultraviolet-visible (UV-vis) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (1H NMR), elemental analysis and electrospray ionization mass spectrometry (ESI-MS) spectra. The [RuCl(qn)(Lbpy2)(NO)]NO3 complex was crystallized and exhibited distorted octahedral geometry, in which the Ru-N(O) bond length was 1.752(6) Å and the Ru-N-O angle was 177.6(6)°. Time-resolved FT-IR and electron paramagnetic resonance (EPR) spectra were used to confirm the photoactivated NO release of the complexes. The binding constant (Kb) of two complexes with human serum albumin (HSA) and DNA were quantitatively evaluated using fluorescence spectroscopy, Ru-Lbpy1 (Kb~106 with HSA and ~104 with DNA) had higher affinity than Ru-Lbpy2. The interactions between the complexes and HSA were investigated using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and EPR spectra. HSA can be used as a carrier to facilitate the release of NO from the complexes upon photoirradiation. The confocal imaging of photo-induced NO release in living cells was successfully observed with a fluorescent NO probe. Moreover, the photocleavage of pBR322 DNA for the complexes and the effect of different Lbpy substituted groups in the complexes on their reactivity were analyzed.


Assuntos
Complexos de Coordenação/química , Substâncias Macromoleculares/química , Rutênio/química , Complexos de Coordenação/síntese química , Complexos de Coordenação/isolamento & purificação , Complexos de Coordenação/farmacologia , DNA/química , DNA/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Ligantes , Substâncias Macromoleculares/ultraestrutura , Óxido Nítrico/biossíntese , Óxido Nítrico/química , Rutênio/farmacologia , Albumina Sérica Humana/química , Espectroscopia de Infravermelho com Transformada de Fourier
4.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916928

RESUMO

Osteoarthritis (OA), a degenerative joint disorder, has been reported as the most common cause of disability worldwide. The production of inflammatory cytokines is the main factor in OA. Previous studies have been reported that obeticholic acid (OCA) and OCA derivatives inhibited the release of proinflammatory cytokines in acute liver failure, but they have not been studied in the progression of OA. In our study, we screened our small synthetic library of OCA derivatives and found T-2054 had anti-inflammatory properties. Meanwhile, the proliferation of RAW 264.7 cells and ATDC5 cells were not affected by T-2054. T-2054 treatment significantly relieved the release of NO, as well as mRNA and protein expression levels of inflammatory cytokines (IL-6, IL-8 and TNF-α) in LPS-induced RAW 264.7 cells. Moreover, T-2054 promoted extracellular matrix (ECM) synthesis in TNF-α-treated ATDC5 chondrocytes. Moreover, T-2054 could relieve the infiltration of inflammatory cells and degeneration of the cartilage matrix and decrease the levels of serum IL-6, IL-8 and TNF-α in DMM-induced C57BL/6 mice models. At the same time, T-2054 showed no obvious toxicity to mice. Mechanistically, T-2054 decreased the extent of p-p65 expression in LPS-induced RAW 264.7 cells and TNF-α-treated ATDC5 chondrocytes. In summary, we showed for the first time that T-2054 effectively reduced the release of inflammatory mediators, as well as promoted extracellular matrix (ECM) synthesis via the NF-κB-signaling pathway. Our findings support the potential use of T-2054 as an effective therapeutic agent for the treatment of OA.


Assuntos
Anti-Inflamatórios/farmacologia , Ácido Quenodesoxicólico/análogos & derivados , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Biomarcadores , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Cartilagem/patologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Quenodesoxicólico/química , Ácido Quenodesoxicólico/farmacologia , Ácido Quenodesoxicólico/uso terapêutico , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Óxido Nítrico/biossíntese , Osteoartrite/tratamento farmacológico , Osteoartrite/etiologia , Osteoartrite/patologia , Células RAW 264.7
5.
Molecules ; 26(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924816

RESUMO

In this paper, chitooligosaccharides in different salt forms, such as chitooligosaccharide lactate, citrate, adipate, etc., were prepared by the microwave method. They were characterized by SEM, FTIR, NMR, etc., and the nitric oxide (NO) expression was determined in RAW 264.7 cells. The results showed that pure chitooligosaccharide was an irregular spherical shape with rough surface, and its different salt type products are amorphous solid with different honeycomb sizes. In addition to the characteristic absorption peaks of chitooligosaccharides, in FTIR, the characteristic absorption of carboxyl group, methylene group, and aromatic group in corresponding acid appeared. The characteristic absorption peaks of carbon in carboxyl group, hydrogen and carbon in methyl, methylene group, and aromatic group in corresponding acid also appeared in NMR. Therefore, the sugar ring structure and linking mode of chitooligosaccharides did not change after salt formation of chitooligosaccharides. Different salt chitooligosaccharides are completely different in promoting NO secretion by macrophages, and pure chitooligosaccharides are the best.


Assuntos
Quitina/análogos & derivados , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Sais/química , Animais , Sobrevivência Celular , Quitina/química , Quitina/farmacologia , Quitina/ultraestrutura , Imageamento por Ressonância Magnética , Camundongos , Estrutura Molecular , Células RAW 264.7 , Espectroscopia de Infravermelho com Transformada de Fourier , Termogravimetria , Difração de Raios X
6.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802652

RESUMO

Red blood cells (RBCs) have been found to synthesize and release both nitric oxide (NO) and cyclic guanosine monophosphate (cGMP), contributing to systemic NO bioavailability. These RBC functions resulted impaired in chronic kidney disease (CKD). This study aimed to evaluate whether predialysis (conservative therapy, CT) and dialysis (peritoneal dialysis, PD; hemodialysis, HD) therapies used during CKD progression may differently affect NO-synthetic pathway in RBCs. Our data demonstrated that compared to PD, although endothelial-NO-synthase activation was similarly increased, HD and CT were associated to cGMP RBCs accumulation, caused by reduced activity of cGMP membrane transporter (MRP4). In parallel, plasma cGMP levels were increased by both CT and HD and they significantly decreased after hemodialysis, suggesting that this might be caused by reduced cGMP renal clearance. As conceivable, compared to healthy subjects, plasma nitrite levels were significantly reduced by HD and CT but not in patients on PD. Additionally, the increased carotid intima-media thickness (IMT) values did not reach the significance exclusively in patients on PD. Therefore, our results show that PD might better preserve the synthetic NO-pathway in CKD-erythrocytes. Whether this translates into a reduced development of uremic vascular complications requires further investigation.


Assuntos
Eritrócitos/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico/sangue , Diálise Peritoneal , Diálise Renal , Uremia/sangue , Idoso , GMP Cíclico/sangue , GMP Cíclico/metabolismo , Feminino , Humanos , Falência Renal Crônica/sangue , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Óxido Nítrico Sintase/metabolismo , Nitritos/sangue , Nitrosação , Fosforilação
7.
J Leukoc Biol ; 109(5): 891-900, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33866588

RESUMO

Myeloid-derived suppressor cells (MDSCs) constitute a heterogeneous population of immature myeloid cells derived from bone marrow and negatively regulate both innate and adaptive immunity in the tumor microenvironment. Previously we have demonstrated that MDSCs lacking histone deacetylase 11 (HDAC11) displayed an increased suppressive activity against CD8+ T-cells. However, the mechanisms of HDAC11 that contribute to the suppressive function of MDSCs remain unclear. Here, we show that arginase activity and NO production is significantly higher in HDAC11 knockout MDSCs when compared with wild-type (WT) controls. In the absence of HDAC11, elevated arginase level and enzymatic activity were observed preferentially in the tumor-infiltrated granulocytic MDSCs, whereas iNOS expression and NO production were increased in the tumor-infiltrated monocytic MDSCs. Of note and for the first time, we demonstrated an association between the elevated expression of immunosuppressive molecules with up-regulation of the transcription factor C/EBPß in MDSCs lacking HDAC11. Interestingly, the highest expression of C/EBPß was observed among CD11b+ Gr-1+ MDSCs isolated from tumor-bearing mice. The additional demonstration that HDAC11 is recruited to the promoter region of C/EBPß in WT MDSCs suggests a novel molecular mechanism by which HDAC11 influence the expression of immunosuppressive molecules in MDSCs through regulation of C/EBPß gene expression.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Células Supressoras Mieloides/imunologia , Animais , Arginase/metabolismo , Células da Medula Óssea/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Deleção de Genes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Regiões Promotoras Genéticas/genética , Regulação para Cima
8.
Mem Inst Oswaldo Cruz ; 116: e200417, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33729328

RESUMO

BACKGROUND: Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-ß signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains. OBJECTIVES: To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole. METHODS: Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence. FINDINGS: Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole. MAIN CONCLUSIONS: These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.


Assuntos
Macrófagos Peritoneais/parasitologia , Macrófagos/parasitologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/biossíntese , Toxoplasma/fisiologia , Animais , Macrófagos/metabolismo , Camundongos , Toxoplasmose Animal/parasitologia
9.
Chem Biodivers ; 18(3): e2000989, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33528898

RESUMO

Artemisia lavandulaefolia, a traditional herbal medicine, has been utilized as anti-inflammatory and analgesia agent in clinic. Bioassay-guided fractionation resulted in a fraction (ALDF) with anti-inflammatory effect obtained from A. lavandulaefolia. Its main constituents were analyzed and identified by UPLC-ESI-Q-TOF-MS technology. ALDF showed the strong inhibitory activity on the nitrogen oxide (NO) production in LPS-induced RAW 264.7 macrophages with an IC50 value of 1.64±0.41 µg/mL. Further results displayed that ALDF also significantly suppressed the secretion of key pro-inflammatory mediators, including tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2 ) and interleukin-1ß (IL-1ß), and the increase of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression induced by LPS stimulation. Mechanism study indicated that ALDF was able to block NF-κB signaling pathway through inhibiting IκB and p65 phosphorylation, as well as NF-κB p65 nuclear translocation. Furthermore, in vivo results in mice revealed that treatments with ALDF evoked significant inhibition on ear edema induced by xylene and on the writhing responses induced by acetic acid. These results suggest that ALDF holds great potential in the prevention and treatment of inflammatory disorders.


Assuntos
Anti-Inflamatórios/farmacologia , Artemisia/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Ácido Acético , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciclo-Oxigenase 2/biossíntese , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/tratamento farmacológico , Feminino , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Dor/induzido quimicamente , Dor/tratamento farmacológico , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Células RAW 264.7 , Estereoisomerismo , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Xilenos
10.
Chem Biodivers ; 18(3): e2001065, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33576170

RESUMO

Two enantiomeric humulane sesquiterpenes, namely (+)-alashanoid N (1a) and (-)-alashanoid N (1b), along with two known analogs ((2R,3R,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one (2) and (2R,3S,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one (3)), were described from the peeled stems of a folk Mongolian herbal medicine Syringa pinnatifolia. Their structures were characterized based on UV, IR, NMR, and HR-ESI-MS data analyses, and the absolute configurations were determined by data analysis of X-ray diffraction and quantum chemical calculations. (+/-)-Alashanoid N showed inhibition against NO production in RAW 264.7 macrophage cells with IC50 values of 90.1 µM and 71.7 µM, and protective effect against oxygen-glucose deprivation injury to H9c2 cells at a concentration of 20 µM and 5 µM, respectively.


Assuntos
Substâncias Protetoras/farmacologia , Syringa/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glucose/antagonistas & inibidores , Glucose/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Conformação Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Oxigênio/metabolismo , Substâncias Protetoras/química , Substâncias Protetoras/isolamento & purificação , Células RAW 264.7 , Ratos , Estereoisomerismo , Relação Estrutura-Atividade
11.
Mol Biol Rep ; 48(3): 2173-2181, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33630206

RESUMO

BACKGROUND: The pawpaw tree has several beneficial effects. However, no studies have been conducted to address the mechanisms underlying the cytotoxic effects of pawpaw extracts against cancer cells, and no study has investigated the anti-inflammatory effects. Hence, in this study, the growth-inhibitory effects of pawpaw (Asimina triloba [L.] Dunal) extracts against gastric (AGS) and cervical (HeLa) cancer cells and the inhibitory effects of pawpaw extracts against inflammatory factors (NO, TNF-α, IL-6, iNOS, and COX-2) were investigated. METHODS AND RESULTS: The viability of AGS and HeLa cells, the analysis of cell cycle, and the expression of apoptosis marker protein were determined using MTT assay, FACS, western blotting, and TUNEL assays. The inflammatory factors were determined using Griess method, ELISA assay kit, and RAW 264.7 cells. The IC50 values of twig and unripe fruit extracts for AGS cells were 82.01 and 100.61 µg/mL, respectively. For HeLa cells, pawpaw twig extracts exhibited the strongest ability to inhibit cervical cancer cell growth (IC50 = 97.73 µg/mL). Analysis of the cell cycle phase distribution and expression of the apoptosis regulatory proteins BCL-2, BAX, caspase-3, and PARP showed that pawpaw twig, root, and unripe fruit extracts induced Sub G1 cell cycle arrest and apoptosis of AGS and HeLa cells. In addition, the twig, root, and unripe fruit extracts of pawpaw effectively inhibited the inflammatory makers NO, TNF-α, IL-6, and iNOS. Particularly, the twig, root, and unripe fruit extracts at concentrations of 50 µg/mL exhibited > 50% inhibition of TNF-α. CONCLUSIONS: These findings indicate that pawpaw extracts are natural therapeutic agents that may be used for the prevention and treatment of gastric and cervical cancers, and encourage further studies on the anti-inflammatory potential of the pawpaw tree.


Assuntos
Anti-Inflamatórios/farmacologia , Asimina/química , Frutas/química , Folhas de Planta/química , Raízes de Plantas/química , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Óxido Nítrico/biossíntese , Células RAW 264.7
12.
Biochem J ; 478(4): 855-869, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33522568

RESUMO

Lupeol is known to be plentiful in fruits or plant barks and has an antimicrobial effect, however, its mode of action(s) has yet to be determined. To elucidate lupeol generates nitric oxide (NO), which is recognized for possessing an antimicrobial activity, intracellular NO was measured in Escherichia coli using DAF-FM. Using the properties of NO passing through plasma membrane easily, increased malondialdehyde levels have shown that lupeol causes lipid peroxidation, and the resulting membrane depolarization was confirmed by DiBAC4(3). These data indicated that lupeol-induced NO is related to the destruction of bacterial membrane. Further study was performed to examine whether NO, known as a cell proliferation inhibitor, affects bacterial cell division. As a result, DAPI staining verified that lupeol promotes cell division arrest, and followed by early apoptosis is observed in Annexin V/PI double staining. Even though these apoptotic hallmarks appeared, the endonuclease failed to perform properly with supporting data of decreased intracellular Mg2+ and Ca2+ levels without DNA fragmentation, which is confirmed using a TUNEL assay. These findings indicated that lupeol-induced NO occurs DNA fragmentation-independent bacterial apoptosis-like death (ALD). Additionally, lupeol triggers DNA filamentation and morphological changes in response to DNA repair system called SOS system. In accordance with the fact that ALD deems to SOS response, and that the RecA is considered as a caspase-like protein, increase in caspase-like protein activation occurred in E. coli wild-type, and no ΔRecA mutant. In conclusion, these results demonstrated that the antibacterial mode of action(s) of lupeol is an ALD while generating NO.


Assuntos
Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Óxido Nítrico/fisiologia , Triterpenos Pentacíclicos/farmacologia , Cálcio/metabolismo , Divisão Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fragmentação do DNA , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Magnésio/metabolismo , Lipídeos de Membrana/metabolismo , Potenciais da Membrana/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/biossíntese , Norfloxacino/farmacologia , Recombinases Rec A/metabolismo , Resposta SOS em Genética
13.
Nutrients ; 13(2)2021 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-33562042

RESUMO

L-Arginine (L-ARG) supplementation has been suggested as a therapeutic option in several diseases, including Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like syndrome (MELAS), arguably the most common mitochondrial disease. It is suggested that L-ARG, a nitric oxide (NO) precursor, can restore NO levels in blood vessels, improving cerebral blood flow. However, NO also participates in mitochondrial processes, such as mitochondrial biogenesis, the regulation of the respiratory chain, and oxidative stress. This study investigated the effects of L-ARG on mitochondrial function, nitric oxide synthesis, and nitro-oxidative stress in cell lines harboring the MELAS mitochondrial DNA (mtDNA) mutation (m.3243A>G). We evaluated mitochondrial enzyme activity, mitochondrial mass, NO concentration, and nitro-oxidative stress. Our results showed that m.3243A>G cells had increased NO levels and protein nitration at basal conditions. Treatment with L-ARG did not affect the mitochondrial function and mass but reduced the intracellular NO concentration and nitrated proteins in m.3243A>G cells. The same treatment led to opposite effects in control cells. In conclusion, we showed that the main effect of L-ARG was on protein nitration. Lowering protein nitration is probably involved in the mechanism related to L-ARG supplementation benefits in MELAS patients.


Assuntos
Arginina/farmacologia , DNA Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular , Humanos , Doenças Mitocondriais/genética , Mutação , Óxido Nítrico/biossíntese
14.
Chem Biodivers ; 18(3): e2000930, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33471368

RESUMO

Enzymatic hydrolysate of the crude polysaccharide (SFP) extracted from Sargassum fusiforme was purified by column DEAE-52 and Sephadex G-100 to yield four components, namely, ESFP1, ESFP2, ESFP3 and ESFP4. These components were characterized by chemical composition assay, GC/MS, HPGPC, UV and FT-IR techniques. The in vitro antioxidant activities of the four purified fractions were investigated by measuring their radical scavenging activity and reducing power. The results suggested that all the four components possess good antioxidant activities. Among them, ESFP1 was found to possess the strongest 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and hydroxyl radical-scavenging activity, and the greatest ferric reducing power. The immunomodulatory effect of these four polysaccharides was demonstrated by their ability to promote proliferation, and to enhance both phagocytic activity and NO release in a macrophage RAW264.7 model. The results revealed that the bioactivities of the polysaccharides are related to their molecular weight, and the uronic acid and sulfate contents.


Assuntos
Antioxidantes/farmacologia , Polissacarídeos/farmacologia , Sargassum/química , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Compostos de Bifenilo/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Estrutura Molecular , Peso Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Picratos/antagonistas & inibidores , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Células RAW 264.7 , Sargassum/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade
15.
Int J Mol Sci ; 22(2)2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33430433

RESUMO

Nitric oxide (NO) is a regulator of growth, development, and stress responses in living organisms. Plant nitrate reductases (NR) catalyze the reduction of nitrate to nitrite or, alternatively, to NO. In plants, NO action and its targets remain incompletely understood, and the way NO regulates its own homeostasis remains to be elucidated. A significant transcriptome overlapping between NO-deficient mutant and NO-treated wild type plants suggests that NO could negatively regulate its biosynthesis. A significant increase in NO content was detected in transgenic plants overexpressing NR1 and NR2 proteins. In turn, NR protein and activity as well as NO content, decreased in wild-type plants exposed to a pulse of NO gas. Tag-aided immunopurification procedures followed by tandem mass spectrometry allowed identifying NO-triggered post-translational modifications (PTMs) and ubiquitylation sites in NRs. Nitration of tyrosine residues and S-nitrosation of cysteine residues affected key amino acids involved in binding the essential FAD and molybdenum cofactors. NO-related PTMs were accompanied by ubiquitylation of lysine residues flanking the nitration and S-nitrosation sites. NO-induced PTMs of NRs potentially inhibit their activities and promote their proteasome-mediated degradation. This auto-regulatory feedback loop may control nitrate assimilation to ammonium and nitrite-derived production of NO under complex environmental conditions.


Assuntos
Homeostase/genética , Nitrato Redutases/genética , Óxido Nítrico/análogos & derivados , Processamento de Proteína Pós-Traducional/genética , Compostos de Amônio/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Taxa de Depuração Metabólica/genética , Nitratos/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico/genética , Nitritos/metabolismo
16.
Chem Pharm Bull (Tokyo) ; 69(1): 99-105, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33390527

RESUMO

A set of isoflavononid and flavonoid analogs was prepared and evaluated for estrogen receptor α (ERα) and ERß transactivation and anti-neuroinflammatory activities. Structure-activity relationship (SAR) study of naturally occurring phytoestrogens, their metabolites, and related isoflavone analogs revealed the importance of the C-ring of isoflavonoids for ER activity and selectivity. Docking study suggested putative binding modes of daidzein 2 and dehydroequol 8 in the active site of ERα and ERß, and provided an understanding of the promising activity and selectivity of dehydroequol 8. Among the tested compounds, equol 7 and dehydroequol 8 were the most potent ERα/ß agonists with ERß selectivity and neuroprotective activity. This study provides knowledge on the SAR of isoflavonoids for further development of potent and selective ER agonists with neuroprotective potential.


Assuntos
Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Fármacos Neuroprotetores/farmacologia , Fitoestrógenos/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Fitoestrógenos/síntese química , Fitoestrógenos/química , Relação Estrutura-Atividade
17.
Parasitol Res ; 120(3): 1025-1035, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33501586

RESUMO

Chicken coccidiosis, caused by an obligate intracellular protozoan parasite of the genus Eimeria, is a major parasitic disease in the intensively reared poultry industry. Due to the widespread use of anticoccidial drugs, resistance has become an inevitable problem. In our previous study, Eimeria tenella citrate synthase (EtCS) was found to be up-expressed in two drug-resistant strains (diclazuril-resistant and maduramycin-resistant strains) compared to drug-sensitive strain by RNA sequence. In this study, we cloned and expressed EtCS and obtain its polyclonal antibodies. Quantitative real-time polymerase chain (qPCR) reactions and Western blots were used to analyze the transcription and translation levels of EtCS in sensitive and three drug-resistant strains. Compared with the sensitive strain, the transcription of EtCS was both significantly upregulated in diclazuril-resistant and maduramycin-resistant strains, but was not significantly different in salinomycin-resistant strain. No significant difference was seen in translation level in the three drug-resistant strains. Indirect immunofluorescence indicated that EtCS was mainly located in the cytoplasm of sporozoites except for posterior refractile bodies and in the cytoplasm and surface of merozoites. Anti-rEtCS antibody has inhibitory effects on E. tenella sporozoite invasion of DF-1 cells and the inhibition rate is more than 83%. Binding of the protein to chicken macrophage (HD11) cells was confirmed by immunofluorescence assays. When macrophages were treated with rEtCS, secretion of nitric oxide and cell proliferation of the macrophages were substantially reduced. These results showed that EtCS may be related to host cell invasion of E. tenella and involve in the development of E.tenella resistance to some drugs.


Assuntos
Galinhas/parasitologia , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Coccidiose/veterinária , Eimeria tenella/enzimologia , Doenças das Aves Domésticas/parasitologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Sequência de Bases , Western Blotting , Citrato (si)-Sintase/imunologia , Citrato (si)-Sintase/isolamento & purificação , Clonagem Molecular , Coccidiose/parasitologia , Eimeria tenella/genética , Eimeria tenella/fisiologia , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Soros Imunes/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Merozoítos/efeitos dos fármacos , Camundongos , Óxido Nítrico/biossíntese , Nitrilas/farmacologia , Piranos/farmacologia , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Organismos Livres de Patógenos Específicos , Esporozoítos/enzimologia , Esporozoítos/imunologia , Triazinas/farmacologia
18.
Int J Biol Macromol ; 172: 550-559, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465362

RESUMO

The crude polysaccharide was extracted from A. asphodeloides rhizomes and further purified to produce two fractions F1 (50.0%) and F2 (19.6%). The chemical constitutions of the polysaccharides were neutral sugars (51.4%-89.7%), uronic acids (1.0%-30.2%) and sulfate esters (3.4%-8.1%), with various ratios of monosaccharides including rhamnose (1.4%-6.1%), arabinose (7.1%-21.2%), xylose (0.2%-4.8%), mannose (39.9%-79.0%), glucose (6.0%-11.1%) and galactose (2.6%-22.0%). The molecular properties of the polysaccharides were investigated by the HPSEC-UV-MALLS-RI system, revealing the Mw 130.0 × 103-576.5 × 103 g/moL, Rg 87.6-382.6 nm and SVg 0.3-54.3 cm3/g. The polysaccharides stimulated RAW264.7 cells to produce considerable amounts of NO and up-regulate the expression of TNF-α, IL-1 and COX-2 genes. Polysaccharides exhibited the growth inhibitory effects on cancer cells lines of AGS, MKN-28 and MKN-45, in which F2 fraction exhibited prominent bioactivities. The AGS cells treated with F2 experienced condensed cytoplasm, shrinkage of nucleus and chromatin marginalization with the highest number of cells at early-stage apoptosis reaching 54.6%. The inhibitory effect of F2 polysaccharide on AGS cells was through MAPKs and STAT3 signaling pathways. The backbone of the F2 was mainly linked by (1 â†’ 4)-linked mannopyranosyl and (1 â†’ 3)-linked galactopyranosyl. Taken together, the polysaccharide from A. asphodeloides rhizomes could be utilized as medicinal, pharmacological and functional food ingredients.


Assuntos
Anemarrhena/química , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Polissacarídeos/farmacologia , Rizoma/química , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Carboidratos , Linhagem Celular Tumoral , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/imunologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Citoplasma/efeitos dos fármacos , Citoplasma/imunologia , Citoplasma/patologia , Fatores Imunológicos/química , Fatores Imunológicos/isolamento & purificação , Interleucina-1/genética , Interleucina-1/imunologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Monossacarídeos/química , Monossacarídeos/isolamento & purificação , Óxido Nítrico/biossíntese , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Células RAW 264.7 , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Ácidos Urônicos/química , Ácidos Urônicos/isolamento & purificação
19.
Eur J Med Chem ; 213: 113162, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33493826

RESUMO

Based on the previous research results of our research group, to further improve the anti-inflammatory activity of hesperetin, we substituted triazole at the 7-OH branch of hesperetin. We also evaluated the anti-inflammatory activity of 39 new hesperetin derivatives. All compounds showed inhibitory effects on nitric oxide (NO) and inflammatory factors in lipopolysaccharide-induced RAW264.7 cells. Compound d5 showed a strong inhibitory effect on NO (half maximal inhibitory concentration = 2.34 ± 0.7 µM) and tumor necrosis factor-α, interleukin (IL)-1ß, and (IL-6). Structure-activity relationships indicate that 7-O-triazole is buried in a medium-sized hydrophobic cavity that binds to the receptor. Compound d5 can also reduce the reactive oxygen species production and significantly inhibit the expression of inducible NO synthase and cyclooxygenase-2 through the nuclear factor-κB signaling pathway. In vivo results indicate that d5 can reduce liver inflammation in mice with acute liver injury (ALI) induced by CCI4. In conclusion, d5 may be a candidate drug for treating inflammation associated with ALI.


Assuntos
Desenho de Fármacos , Hesperidina/farmacologia , Inflamação/tratamento farmacológico , Fígado/efeitos dos fármacos , Triazóis/farmacologia , Animais , Tetracloreto de Carbono , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Hesperidina/síntese química , Hesperidina/química , Inflamação/metabolismo , Inflamação/patologia , Fígado/lesões , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Óxido Nítrico/análise , Óxido Nítrico/biossíntese , Células RAW 264.7 , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química
20.
Eur J Med Chem ; 213: 113174, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33515864

RESUMO

In order to discover and develop drug-like anti-inflammatory agents against arthritis, based on "Hit" we found earlier and to overcome drawbacks of toxicity, twelve series of total 89 novel pyrimidine, pyrazolo[4,3-d]pyrimidine and thieno[3,2-d]pyrimidine derivatives were designed, synthesized and screened for their anti-inflammatory activity against NO and toxicity for normal liver cells (LO2). Relationships of balance toxicity and activity have been summarized through multi-steps, and title compounds 22o, 22l were found to show lower toxicity (against LO2: IC50 = 2934, 2301 µM, respectively) and potent effect against NO release (IR = 98.3, 97.67%, at 10 µM, respectively). Furthermore, compound 22o showed potent iNOS inhibitory activity with value of IC50 is 0.96 µM and could interfere stability and formation of the active dimeric iNOS. It's anti-inflammatory activity in vivo was assessed by AIA rat model. Furthermore, the results of metabolic stability, CYP, PK study in vivo, acute toxicity study and subacute toxicity assessment indicated this compound had good drug-like properties for treatment.


Assuntos
Artrite/tratamento farmacológico , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Administração Oral , Animais , Artrite/metabolismo , Células Cultivadas , Dimerização , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Adjuvante de Freund , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Estrutura Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Pirazóis/administração & dosagem , Pirazóis/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Células RAW 264.7 , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade
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