RESUMEN
One of the important monitoring indicators of the air pollution is atmospheric fine particulate matter (PM2.5 ), which can induce lung inflammation after inhalation. Coelonin can alleviate PM2.5 -induced macrophage damage through anti-inflammation. However, its molecular mechanism remains unclear. We hypothesized that macrophage damage may involve the release of inflammatory cytokines, activation of inflammatory pathways, and pyrosis induced by inflammasome. In this study, we evaluated the anti-inflammation activity of coelonin in PM2.5 -induced macrophage and its mechanism of action. Nitric oxide (NO) and reactive oxygen species (ROS) production were measured by NO Assay kit and dichlorofluorescein-diacetate (DCFH-DA), and apoptosis were measured by Flow cytometry and TUNEL staining. The concentration of inflammatory cytokines production was measured with cytometric bead arrays and ELISA kits. The activation of NF-κB signaling pathway and NLRP3 inflammasome were measured by immunofluorescence, quantitative reverse transcription-polymerase chain reaction and western blot. As expected, coelonin pretreatment reduced NO production significantly as well as alleviated cell damage by decreasing ROS and apoptosis. It decreased generation of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in PM2.5 -induced RAW264.7 and J774A.1 cells. Moreover, coelonin markedly inhibited upregulating the expression of toll-like receptor (TLR)4 and cyclo-oxygenase (COX)-2, blocked activation of p-nuclear factor-kappa B (NF-κB) signaling pathway, and suppressed expression of NLRP3 inflammasome, ASC, GSDMD, IL-18 and IL-1ß. In conclusion, the results showed that coelonin could protect against PM2.5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.
Asunto(s)
Inflamasomas , FN-kappa B , FN-kappa B/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ciclooxigenasa 2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/metabolismo , Transducción de Señal , Macrófagos/metabolismo , Citocinas/metabolismo , Interleucina-6 , Antiinflamatorios/farmacología , Material Particulado/toxicidadRESUMEN
Certain mutations in mitochondrial DNA (mtDNA) are associated with Leber's hereditary optic neuropathy (LHON). In particular, the wellknown NADH dehydrogenase 4 (ND4) m.11778G>A mutation is one of the most common LHONassociated primary mutations worldwide. However, how specific mtDNA mutations, or variants, affect LHON penetrance is not fully understood. The aim of the current study was to explore the relationship between mtDNA mutations and LHON, and to provide useful information for early detection and prevention of this disease. Following the molecular characterization of a Han Chinese family with maternally inherited LHON, four out of eight matrilineal relatives demonstrated varying degrees of both visual impairment and age of onset. Through PCR amplification of mitochondrial genomes and direct Sanger sequencing analysis, a homoplasmic mitochondrialencoded ND4 m.11778G>A mutation, alongside a set of genetic variations belonging to human mtDNA haplogroup B5b1 were identified. Among these sequence variants, alanine transfer RNA (tRNA)Ala m.5601C>T was of particular interest. This variant occurred at position 59 in the TψC loop and altered the base pairing, which led to mitochondrial RNA (mtRNA) metabolism failure and defects in mitochondrial protein synthesis. Bioinformatics analysis suggested that the m.5601C>T variant altered tRNAAla structure. Therefore, impaired mitochondrial functions caused by the ND4 m.11778G>A mutation may be enhanced by the mttRNAAla m.5601C>T variant. These findings suggested that the tRNAAla m.5601C>T variant might modulate the clinical manifestation of the LHONassociated primary mutation.
Asunto(s)
ADN Mitocondrial/genética , Mitocondrias/genética , NADH Deshidrogenasa/genética , Atrofia Óptica Hereditaria de Leber/genética , ARN de Transferencia de Alanina/genética , Adolescente , Adulto , Pueblo Asiatico/genética , Niño , Biología Computacional , Familia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Atrofia Óptica Hereditaria de Leber/sangre , Atrofia Óptica Hereditaria de Leber/metabolismo , Atrofia Óptica Hereditaria de Leber/patología , Linaje , Penetrancia , Filogenia , Polimorfismo Genético , ARN de Transferencia de Alanina/químicaRESUMEN
BACKGROUND: Bletilla striata is a traditional Chinese medicine used to treat hemorrhage, scald, gastric ulcer, pulmonary diseases and inflammations. In this study, we investigated bioactivity of the effective fraction of B. striata (EFB) in reducing the inflammatory cytokine production induced by water or organic extracts of PM2.5. METHODS: PM2.5 extracts were collected and analyzed by chromatographic system and inductively coupled plasma mass spectrometer. Cell viability was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, and cell supernatant was analyzed by flow cytometry, ELISA, and qRT-PCR in cultured mouse macrophage cell line RAW264.7 treated with EFB and PM2.5 extracts. Expressions of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway were measured by Western blot. RESULTS: PM2.5 composition is complex and the toxicity of PM2.5 extracts were not noticeable. The treatment of EFB at a wide dose-range of 0-40 µg/mL did not cause significant change of RAW264.7 cell proliferation. EFB pretreatment decreased the inflammatory cytokines in the macrophage. Further analysis showed that EFB significantly attenuated PM2.5-induced proinflammatory protein expression and downregulated the levels of phosphorylated NF-κBp65, inhibitor of kappa B (IκB)-α, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38. CONCLUSIONS: Our study demonstrated the potential effectiveness of B. striata extracts for treating PM2.5-triggered pulmonary inflammation.
Asunto(s)
Antiinflamatorios/farmacología , Citocinas/metabolismo , Orchidaceae , Material Particulado/toxicidad , Extractos Vegetales/farmacología , Animales , Antiinflamatorios/química , Supervivencia Celular/efectos de los fármacos , Citocinas/análisis , Citocinas/genética , Inflamación/metabolismo , Ratones , Modelos Inmunológicos , Extractos Vegetales/química , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
In order to understand the difference of contents of coelonin,batatasin â ¢ and 3'-O-methylbatatasin â ¢ in 60 different sources of Bletilla striata planted under the same conditions. UPLC method was used and the analysis was performed on a Waters ACQUITY UPLC BEH C18 column( 2. 1 mm×100 mm,1. 7 µm),eluted with acetonitril-0. 1% formic acid solution by gradient. The flow rate was 0. 208 m L·min-1,the detection wavelength was 270 nm,the column temperature was 35 â and the injection volume was 4µL. Under the above chromatographic conditions,the three components can be separated well with good linearity in the range of 0. 156-5. 000 mg·L-1. The average contents of coelonin,batatasin â ¢ and 3'-O-methylbatatasin â ¢ were( 0. 116 ± 0. 071) %,( 0. 386 ±0. 185) % and( 0. 086±0. 034) %,respectively. After planting for two years under the same conditions,there was no significant difference in chemical composition among different sources and varieties,but the contents of the three components had some regional differences,which indicated that the western region was higher than the eastern region,while the contents of coelonin and batatasin â ¢ in B.sinensis were slightly higher than those in B. striata. The chromatographic method above is simple,stable and reproducible,and can be used for quantitative analysis of three components. The content analysis of different sources of B. striata can provide reference for future B. striata breeding and quality control.
Asunto(s)
Medicamentos Herbarios Chinos/química , Orchidaceae/química , Cromatografía Líquida de Alta Presión , Control de CalidadRESUMEN
Mutations in mitochondrial 12S rRNA (MT-RNR1) are the important causes of sensorineural hearing loss. Of these mutations, the homoplasmic m.1555A>G or m.1494C>T mutation in the highly conserved A-site of MT-RNR1 gene has been found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. Since the m.1555A>G and m.1494C>T mutations are sensitive to ototoxic drugs, therefore, screening for the presence of these mutations is important for early diagnosis and prevention of deafness. For this purpose, we recently developed a novel allele-specific PCR (AS-PCR) which is able to simultaneously detect these mutations. To assess its accuracy, in this study, we employed this method to screen the frequency of m.1555A>G and m.1494C>T mutations in 200 deafness patients and 120 healthy subjects. Consequently, four m.1555A>G and four m.1494C>T mutations were identified; among these, only one patient with the m.1494C>T mutation had an obvious family history of hearing loss. Strikingly, clinical evaluation showed that this family exhibited a high penetrance of hearing loss. In particular, the penetrances of hearing loss were 80% with the aminoglycoside included and 20% when excluded. PCR-Sanger sequencing of the mitochondrial genomes confirmed the presence of the m.1494C>T mutation and identified a set of polymorphisms belonging to mitochondrial haplogroup A. However, the lack of functional variants in mitochondrial and nuclear modified genes (GJB2 and TRMU) in this family indicated that mitochondrial haplogroup and nuclear genes may not play important roles in the phenotypic expression of the m.1494C>T mutation. Thus, other modification factors, such as environmental factor, aminoglycosides or epigenetic modification may have contributed to the high penetrance of hearing loss in this family. Taken together, our data showed that this assay is an effective approach that could be used for detection the deafness-associated MT-RNR1 mutations.
Asunto(s)
Alelos , Sordera/genética , Mitocondrias/genética , Mutación/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico/genética , Adulto , Anciano , Pueblo Asiatico/genética , Audiometría , Secuencia de Bases , Conexina 26 , Conexinas/genética , Análisis Mutacional de ADN , Etnicidad/genética , Femenino , Genoma Mitocondrial , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Linaje , ARNt Metiltransferasas/genéticaRESUMEN
Objective: To investigate the anti-pulmonary fibrosis effect and the possible molecular mechanism of the Bletilla striata polysaccharide. Methods: Polysaccharide was prepared by water reflux extraction plus ethanol precipitation method, and following deproteinization process by Sevage method. Rat silicosis model was established by invasive intratracheal instillation method. The effect and molecular mechanism of the polysaccharide was evaluated by lung indexes, lung pathological change, serum levels of SOD,MDA,NF-κB,IL-1ß,PDGF,TGF-ß1,TNF-α,HYP were detected, and the contents of CD3~+,CD4~+,CD8~+T lymph cells and CD4~+/ CD8~+ratio were detected by flow cytometry. Results: Both low( 100 mg / kg) and high( 400 mg / kg) dosage polysaccharide treatment could remarkably elevate the serum SOD level and reduce the MDA,NO level, and effectively reverse the CD4~+/ CD8~+ratio comparing with the model group( P < 0. 01). Except the TNF-α level was significantly lower in the high dosage treatment group, there was no other effect in inflammatory cytokines and HYP content in serum. HE pathological section confirmed that the Bletilla striata polysaccharide treatment group can not effectively prevent lung fibrosis. Conclusion: The Bletilla striata polysaccharide has remarkable regulation effect on antioxidation system and immune system, but can not effectively prevent lung fibrosis, more effort should be made to study the active antipulmonary fibrosis components of Bletilla striata.
Asunto(s)
Fibrosis Pulmonar , Silicosis , Animales , Antioxidantes , Interleucina-1beta , Pulmón , FN-kappa B , Orchidaceae , Extractos Vegetales , Polisacáridos , Ratas , Factor de Crecimiento Transformador beta1 , Factor de Necrosis Tumoral alfaRESUMEN
Objective: To investigate the chemical constituents isolated from the fibrous roots of Bletilla striata, and to research their antibacterial activities. Methods: The native products were isolated and purified by silica gel, Sephadex LH-20 column chromatography and preparative HPLC. Their structures were elucidated on the basis of various spectroscopic analysis, and their antibacterial activities were tested by microbroth dilution method in a 96-well microtiter plate. Results: Seven compounds were isolated from the ethanol extract of the fibrous roots of Bletilla striata, and identified as p-hydroxybenzaldehyde( 1),2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene( 2),4,5-dihydroxy-2-methoxy-9,10-dihydrohenanthrpene( 3),2-dihydroxy-4,7-dimethoxyphenan-threne( 4), militarine( 5), dactylorhin A( 6) and gastrodin( 7). Among them, compounds 2 ~ 4 showed moderate antibacterial activities against several Gram-positive bacterial strains( MIC 8 ~ 128 µg / m L),such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Bacillus subtilis. Conclusion: The fibrous roots and tubers of Bletilla striata contain similar compounds, including glucosyloxybenzyl 2-isobutylmalates,and phenanthrene compounds, which showed antimicrobial activities against Gram-positive bacterial strains. And compounds 3,4 are isolated from Bletilla genus for the first time.