RESUMO
Corylifol A (CA), a phenolic compound from Psoralea corylifolia, possessed several biological properties but poor bioavailability. Here we aimed to investigate the roles of cytochromes P450s (CYPs), UDP-glucuronosyltransferases (UGTs) and efflux transporters in metabolism and disposition of CA.Metabolism of CA was evaluated in HLM, expressed CYPs and UGTs. Chemical inhibitors and shRNA-mediated gene silencing of multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP) were performed to assess the roles of transporters in CA disposition.Three oxidated metabolites (M1-M3) and two glucuronides (M4-M5) were detected. The intrinsic clearances (CLint) values of M1 and M4 in HLM were 48.10 and 184.03 µL/min/mg, respectively. Additionally, CYP1A1, 2C8 and 2C19 were identified as main contributors with CLint values of 13.01-49.36 µL/min/mg, while UGT1A1, 1A7, 1A8 and 1A9 were with CLint values ranging from 85.01 to 284.07 µL/min/mg. Furthermore, activity correlation analysis proved CYP2C8, UGT1A1 and 1A9 were the main active hepatic isozymes. Besides, rats and monkeys were appropriate model animals. Moreover, dipyridamole and MK571 both could significantly inhibit M4 efflux. Gene silencing results also indicated MRP4 and BCRP were major contributors in HeLa1A1 cells.Taken together, CYPs, UGTs, MRP4 and BCRP were important determinants of CA pharmacokinetics.
Assuntos
Flavonas/metabolismo , Animais , Transporte Biológico , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Células HeLa , Humanos , Psoralea , RatosRESUMO
BACKGROUND: Targeting the costimulatory receptor CD137 has shown promise as a therapeutic approach for cancer immunotherapy, resulting in anti-tumor efficacy demonstrated in clinical trials. However, the initial CD137 agonistic antibodies, urelumab and utomilumab, faced challenges in clinical trials due to the liver toxicity or lack of efficacy, respectively. Concurrently, c-MET has been identified as a highly expressed tumor-associated antigen (TAA) in various solid and soft tumors. METHODS: In this study, we aimed to develop a bispecific antibody (BsAb) that targets both c-MET and CD137, optimizing the BsAb format and CD137 binder for efficient delivery of the CD137 agonist to the tumor microenvironment (TME). We employed a monovalent c-MET motif and a trimeric CD137 Variable Heavy domain of Heavy chain (VHH) for the BsAb design. RESULTS: Our results demonstrate that the c-MET x CD137 BsAb provides co-stimulation to T cells through cross-linking by c-MET-expressing tumor cells. Functional immune assays confirmed the enhanced efficacy and potency of the c-MET x CD137 BsAb, as indicated by activation of CD137 signaling, target cell killing, and cytokine release in various tumor cell lines. Furthermore, the combination of c-MET x CD137 BsAb with Pembrolizumab showed a dose-dependent enhancement of target-induced T cell cytokine release. CONCLUSION: Overall, the c-MET x CD137 BsAb exhibits a promising developability profile as a tumor-targeted immune agonist by minimizing off-target effects while effectively delivering immune agonism. It has the potential to overcome resistance to anti-PD-(L)1 therapies.
Assuntos
Anticorpos Biespecíficos , Imunoterapia , Proteínas Proto-Oncogênicas c-met , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , Animais , Humanos , Camundongos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Proto-Oncogênicas c-met/imunologia , Microambiente Tumoral/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
A near-infrared fluorescent probe (TX-P) for detecting peroxynitrite is constructed. The probe has a near-infrared emission (725 nm), large Stokes shift (125 nm) and excellent sensitivity and selectivity. In addition, TX-P can be used to visualize ONOO- in living cells, image ONOO- in paw edema mice and evaluate anti-inflammatory drugs.
Assuntos
Edema , Corantes Fluorescentes , Ácido Peroxinitroso , Animais , Ácido Peroxinitroso/metabolismo , Ácido Peroxinitroso/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Camundongos , Edema/diagnóstico por imagem , Edema/tratamento farmacológico , Edema/induzido quimicamente , Raios Infravermelhos , Humanos , Imagem Óptica , Células RAW 264.7 , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/uso terapêuticoRESUMO
BACKGROUND: Glutathione (GSH), a highly abundant thiol compound within cells, plays a critical role in physiological processes and exhibits close correlation with cancer. Among molecular imaging technologies, most probes have relatively short emission wavelengths and lack photoacoustic imaging (PA) capability, resulting in the inability to obtain tissue images with high penetration depth. The presence of GSH in the tumor microenvironment neutralizes ROS, diminishing the therapeutic effect of PDT, thus resulting in often unsatisfactory therapeutic efficacy. Therefore, it is imperative to develop a dual-modal probe for the detection of GSH and the diagnosis and treatment of cancer. RESULTS: In this study, we synthesized a novel dual-modal probe, Cy-Bio-GSH, utilizing near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging techniques for GSH detection. The probe integrates cyanine dye as the fluorophore, nitroazobenzene as the recognition moiety, and biotin as the tumor-targeting moiety. Upon reacting with GSH, the probe emits NIR fluorescence at 820 nm and generates a PA signal. Significantly, this reaction activates the photodynamic and photothermal properties of the probe. By depleting GSH and employing a synergistic photothermal therapy (PTT) treatment, the therapeutic efficacy of photodynamic therapy (PDT) is remarkably enhanced. In-vivo experiments confirm the capability of the probe to detect GSH via NIRF and PA imaging. Notably, the combined tumor-targeting ability and PDT/PTT synergistic therapy enhance therapeutic outcomes for tumors and facilitate their ablation. SIGNIFICANCE: A novel tumor-targeting and dual-modal imaging probe (Cy-Bio-GSH) is synthesized, exhibiting remarkable sensitivity and selectivity to GSH, enabling the visualization of GSH in cells and the differentiation between normal and cancer cells. Cy-Bio-GSH enhances PDT/PTT with effective killing of cancer cells and makes the ablation of tumors in mice. This work represents the first tumor-targeting probe for GSH detection, and provides crucial tool for cancer diagnosis and treatment by dual-modal imaging with improved PDT/PTT synergistic therapy.
Assuntos
Biotina , Glutationa , Técnicas Fotoacústicas , Fotoquimioterapia , Glutationa/química , Glutationa/metabolismo , Animais , Humanos , Camundongos , Biotina/química , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Imagem Óptica , Feminino , Terapia Fototérmica , Camundongos Nus , Camundongos Endogâmicos BALB C , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/uso terapêuticoRESUMO
BACKGROUND: The proposal of Q-markers for traditional Chinese medicine (TCM) represents a novel avenue of research pertaining to the quality control of TCM prescriptions. However, prior exploratory studies on Q-markers with multiple properties consistently neglected the consideration of weights, hampering our ability to accurately gauge the significance of each property and potentially leading to a flawed comprehension of Q-markers. PURPOSE: In this study, a quantitative ternary network strategy was firstly proposed to visually discover the Q-markers from TCM prescriptions, and it has been successfully applied into the quality control study of Bu-Zhong-Yi-Qi-Tang (BZYQT), a classical TCM prescription. METHODS: Firstly, the contents of 34 components in BZYQT, along with the kinetic features of 17 candidate Q-markers in biosamples (plasma and small intestinal contents), were characterized by UPLC-QqQ-MS/MS, and their immunomodulatory activities in macrophages and splenic lymphocytes were also assessed. Next, the obtained data were integrated into three properties: testability, bioavailability, effectiveness, and their weights were calculated using the entropy weight method to further establish a ternary network for quantitatively screening Q-markers. Subsequently, the identified Q-markers of BZYQT were utilized for the holistic quality evaluation of 36 batches of the commercial BZYQT preparation, Bu-Zhong-Yi-Qi-Pill (BZYQP) produced by three manufacturers, through similarity evaluation of the Q-marker-based fingerprint. RESULTS: Nine compounds (hesperidin, astragaloside IV, ononin, 18ß-glycyrrhizic acid, narirutin, calycosin, cimigenoside, astragaloside II, and liquiritin) showing three core properties, including testability, bioavailability, and effectiveness, were screened out as Q-markers of BZYQT based on their rankings in terms of regression area of the ternary network. Employing Q-markers as common peaks, the similarity values of 36 batches BZYQP ranged 0.914-0.998 under HPLC-UVD mode, and 0.631-1.000 under HPLC-ELSD mode, which were less than the similarity values evaluated by the conventional common peaks (HPLC-UVD mode: 0.946-0.990; HPLC-ELSD mode: 0.957-0.997). This observation suggests that the identified Q-markers are more representative as common peaks in chromatographic fingerprints for the holistic quality evaluation of TCM-related products from different manufacturers. CONCLUSION: The quantitative discovery of Q-markers from BZYQT laid an important foundation for holistic quality assessment of its related commercially available products, and our work offering a new strategy for ensuring the consistency and efficacy of TCM prescriptions.
Assuntos
Medicamentos de Ervas Chinesas , Medicina Tradicional Chinesa , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/análise , Animais , Cromatografia Líquida de Alta Pressão/métodos , Controle de Qualidade , Espectrometria de Massas em Tandem/métodos , Masculino , CamundongosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Bu-Zhong-Yi-Qi-Tang is a famous traditional Chinese medicine formula that has been prevalent in China for over 700 years to treat spleen-qi deficiency related diseases, such as gastrointestinal and respiratory disorders. However, the bioactive components responsible for regulating spleen-qi deficiency remain unclear and have puzzled many researchers. AIM OF THE STUDY: The current study focuses on efficacy evaluation of regulating spleen-qi deficiency and screening the bioactive components of Bu-Zhong-Yi-Qi-Tang. MATERIALS AND METHODS: The effects of Bu-Zhong-Yi-Qi-Tang were evaluated through blood routine examination, immune organ index, and biochemical analysis. Metabolomics was utilized to analyze the potential endogenous biomarkers (endobiotics) in the plasma, and the prototypes (xenobiotics) of Bu-Zhong-Yi-Qi-Tang in the bio-samples were characterized using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Then, these endobiotics were used as "bait" to predict targets based on network pharmacology and to screen potential bioactive components from the absorbed prototypes in the plasma by constructing an "endobiotics-targets-xenobiotics" association network. Further, the anti-inflammatory activities of representative compounds (calycosin and nobiletin) were validated through poly(I:C)-induced pulmonary inflammation mice model. RESULTS: Bu-Zhong-Yi-Qi-Tang exhibited immunomodulatory and anti-inflammatory activities in spleen-qi deficiency rat, as supported by the observation of increased levels of D-xylose and gastrin in serum, an increase in the thymus index and number of lymphocytes in blood, as well as a reduction in the level of IL-6 in bronchoalveolar lavage fluid. Furthermore, plasma metabolomic analysis revealed a total of 36 Bu-Zhong-Yi-Qi-Tang related endobiotics, which were mainly enriched in primary bile acids biosynthesis, the metabolism of linoleic acid, and the metabolism of phenylalanine pathways. Meanwhile, 95 xenobiotics were characterized in plasma, urine, small intestinal contents, and tissues of spleen-qi deficiency rat after Bu-Zhong-Yi-Qi-Tang treatment. Using an integrated association network, six potential bioactive components of Bu-Zhong-Yi-Qi-Tang were screened. Among them, calycosin was found to significantly reduce the levels of IL-6 and TNF-α in the bronchoalveolar lavage fluid, increase the number of lymphocytes, while nobiletin dramatically decreased the levels of CXCL10, TNF-α, GM-CSF, and IL-6. CONCLUSION: Our study proposed an available strategy for screening bioactive components of BYZQT regulating spleen-qi deficiency based on "endobiotics-targets-xenobiotics" association network.
Assuntos
Medicamentos de Ervas Chinesas , Baço , Camundongos , Ratos , Animais , Fator de Necrose Tumoral alfa/farmacologia , Interleucina-6 , Xenobióticos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Medicamentos de Ervas Chinesas/química , Anti-Inflamatórios/farmacologiaRESUMO
Component overlapping and long-time consumption hinder the data processing of offline two-dimensional liquid chromatography mass spectrometry (offline 2D-LC MS) system. Although molecular networking has been commonly employed in data processing of liquid chromatography mass spectrometry (LC-MS), its application in offline 2D-LC MS is challenged by voluminous and redundant data. In light of this, for the first time, a data deduplication and visualization strategy combining hand-in-hand alignment with targeted molecular networking (TMN) for compounds annotation of offline 2D-LC MS data was developed and applied to the chemical profile of Yupingfeng (YPF), a classical traditional Chinese medicine (TCM) prescription, as a case study. Firstly, an offline 2D-LC MS system was constructed for the separation and data acquisition of YPF extract. Then the data of 12 fractions derived from YPF were deconvoluted and aligned as a whole data file by hand-in-hand alignment, resulting in a 49.2% reduction in component overlapping (from 17951 to 9112 ions) and an improvement in the MS2 spectrum quality of precursor ions. Subsequently, the MS2-similarity adjacency matrix of focused parent ions was computed by a self-building Python script, which realized the construction of an innovative TMN. Interestingly, the TMN was found to be able to efficiently distinguish and visualize the co-elution, in-source fragmentations and multi-type adduct ions in a clustering network. Consequently, a total of 497 compounds were successfully identified depending on only seven TMN analysis guided by product ions filtering (PIF) and neutral loss filtering (NLF) for the targeted compounds in YPF. This integrated strategy improved the efficiency of targeted compound discovery in offline 2D-LC MS data, also shown a huge scalability in accurate compound annotation of complex samples. In conclusion, our study developed available concepts and tools while providing a research paradigm for efficient and rapid compound annotation in complex samples such as TCM prescriptions, with YPF as an example.
Assuntos
Medicamentos de Ervas Chinesas , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas , Medicamentos de Ervas Chinesas/químicaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Coronary heart disease (CHD), one of the leading causes of mortality in the world among chronic non-infectious diseases, is closely associated with atherosclerosis, which ultimately leads to myocardial injury. Wendan decoction (WDD), a classical famous formula, exerted an intervention effect on CHD according to numerous reports. However, the effective components and underlying mechanisms for the treatment of CHD have not been fully elucidated. AIM OF THE STUDY: An in-depth investigation of the effective components and mechanisms of WDD for the intervention of CHD was further explored. MATERIALS AND METHODS: Firstly, based on our previous metabolic profile results, a quantification method for absorbed components was established by ultra-performance liquid chromatography triple quadrupole-mass spectrometry (UPLC-TQ-MS) and applied to the pharmacokinetics study of WDD. Then the network pharmacology analysis for considerable exposure components in rat plasma was employed to screen key components of WDD. Gene ontology and KEGG pathway enrichment analysis were further performed to obtain putative action pathways. The effective components and mechanism of WDD were confirmed by in vitro experiments. RESULTS: A rapid and sensitive quantification method was successfully applied to the pharmacokinetic study of 16 high-exposure components of WDD at three different doses. A total of 235 putative CHD targets were obtained for these 16 components. Then, 44 core targets and 10 key components with high degree values were successively screened out by the investigation of protein-protein interaction and the network of "herbal medicine-key components-core targets". Enrichment analysis suggested that the PI3K-Akt signaling pathway was closely related to this formula's therapeutic mechanism. Furthermore, pharmacological experiments demonstrated that 5 of 10 key components (liquiritigenin, narigenin, hesperetin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and isoliquiritigenin) significantly enhanced DOX-induced H9c2 cell viability. The cardioprotective effects of WDD against DOX-induced cell death through the PI3K-Akt signaling pathway were verified by western blot experiments. CONCLUSION: The integration of pharmacokinetics and network pharmacology approaches successfully clarified 5 effective components and therapeutic mechanism of WDD for the intervention of CHD.
Assuntos
Doença das Coronárias , Medicamentos de Ervas Chinesas , Animais , Ratos , Farmacologia em Rede , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Doença das Coronárias/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Simulação de Acoplamento MolecularRESUMO
Pulmonary inflammation caused by respiratory tract viral infections is usually associated with acute exacerbation of respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Therefore, maintaining the pulmonary immune homeostasis is particular important for prevention of the acute exacerbation. Bu-Zhong-Yi-Qi-Tang (BZYQT), a traditional Chinese medicine formula, has been broadly used to improve respiratory and gastrointestinal disorders in China for over 700 years. Previously, we have found the regulatory activity of BZYQT on the lower respiratory immune system, while its potential effects during pulmonary inflammation remain unknown. Thus, the current study focused on deciphering its immunomodulatory effect and potential mechanism against pulmonary inflammation by using a viral RNA analogue, poly (I:C), induced murine pulmonary inflammation model and BEAS-2B cell model coupled with network pharmacology. Inflammatory cells in the bronchoalveolar lavage fluid were counted through microscope examination according to the cell's morphology and staining characteristics; protein and gene levels of inflammatory mediators were determined with Elisa and quantitative PCR, respectively; network pharmacology was conducted based on 46 BZYQT-related potential bioactive components, pulmonary inflammation and immune-related targets. Our results indicated that the recruitment of neutrophils and the expression of Adgre1 (encoding the F4/80, which is a macrophage marker) in the lung induced by poly (I:C) were significantly reduced after BZYQT treatment, and these effects were further demonstrated to be related to the interference of leukocyte transendothelial migration from the decreased levels of CXCL10, IL-6, TNF-α, CXCL2, ICAM-1, VCAM-1, and E/P-selectins. Furthermore, BZYQT inhibited the CXCL10, TNF-α, and IFN-ß expression of poly (I:C)-challenged BEAS-2B cells in a dose-dependent manner. Through integrating results from network pharmacology, experiments, and the published literature, isoliquiritigenin, Z-ligustilide, atractylenolide I, atractylenolide III, formononetin, ferulic acid, hesperidin, and cimigenoside were presumed as the bioactive components of BZYQT against pulmonary inflammation. Overall, our findings demonstrated that BZYQT possesses a pronounced immunomodulatory effect on poly (I:C)-induced pulmonary inflammation, which provides a pharmacological basis for BZYQT in the treatment of respiratory disorders.
RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The dried rhizome of Cimicifuga heracleifolia Kom. (C. heracleifolia) is a popular traditional Chinese medicine, which has been extensively used in Asian countries for its anti-inflammatory, antipyretic and analgesic activities. However, further utilization and application of C. heracleifolia have been hampered due to a lack of full understanding of its active ingredients. AIM OF STUDY: The present study aims for clarification of the systematical chemical profile of C. heracleifolia and the immunomodulatory effect of its main bioavailable component. MATERIALS AND METHODS: Comprehensive chemical profile of C. heracleifolia was systematically analyzed by ultra-performance liquid chromatography hyphenated with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS). Xenobiotics after oral administration of C. heracleifolia extracts were investigated to hunt for bioavailable components. The immunomodulatory activity evaluation of cimigenoside was achieved on poly(I:C)-induced airway inflammation mouse and BEAS-2B cell models from aspects of neutrophil infiltration, lung inflammation by using microscope analysis, quantification of production and expression of inflammatory cytokine and chemokines by using ELISA and quantitative PCR. RESULTS: By UPLC-Q-TOF/MS analysis, 110 compounds (including 81 triterpenoids, 21 cinnamic acid derivatives, and 8 other structure types) were identified or tentatively characterized in ethanolic extract of C. heracleifolia. Based on the data of chemical profile, xenobiotics of C. heracleifolia were subsequently analyzed, and triterpene glycosides were detected as the major bioavailable ingredients. Oral administration of cimigenoside, a representative triterpene glycoside, could prevent neutrophils infiltration in the lung due to suppression of the production of CXCL2 and CXCL10, and the expression of P-selectin, VCAM1 in poly(I:C)-induced airway inflammation model mice. Moreover, cimigenoside also inhibited the productions of inflammatory cytokines and chemokines from human airway epithelial cell line (BEAS-2B cells) induced by poly(I:C). CONCLUSION: Triterpene glycosides were the main components of C. heracleifolia extract, and cimigenoside was considered as the effective component with immunomodulatory effect on the pulmonary immune system by oral administration.
Assuntos
Anti-Inflamatórios/farmacologia , Cimicifuga , Fatores Imunológicos/farmacologia , Pulmão/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pneumonia/prevenção & controle , Animais , Anti-Inflamatórios/isolamento & purificação , Linhagem Celular , Cimicifuga/química , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Fatores Imunológicos/isolamento & purificação , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Pneumonia/induzido quimicamente , Pneumonia/imunologia , Pneumonia/metabolismo , Poli I-C , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
OBJECTIVES: Bavachin is a bioactive natural flavonoid with oestrogen-like activity. Here, we aimed to investigate its metabolic and disposal fates involving in CYPs, UGTs and efflux transporters. METHODS: Phase I metabolism and glucuronidation were performed by human liver microsomes (HLM). Reaction phenotyping and activity correlation analysis were performed to identify the main CYP and UGT isozymes. Chemical inhibition and gene knock-down approaches were employed to explore the function of BCRP and MRPs. KEY FINDINGS: Five phase I metabolites (M1-M5) and three glucuronides (G1-G3) were identified. The CLint values for M4 and G1 by HLM were 127.99 and 1159.07 µl/min per mg, respectively. Reaction phenotyping results suggested CYP1A1 (208.85 µl/min per mg) and CYP2C9 (107.51 µl/min per mg), and UGT1A1 (697.19 µl/min per mg), UGT1A7 (535.78 µl/min per mg), UGT1A8 (247.72 µl/min per mg) and UGT1A9 (783.68 µl/min per mg) all participated in the metabolism of bavachin. In addition, activity correlation analysis also supported the results above. Furthermore, the metabolism exhibited marked species differences, and rabbits were the appropriate model animals. Moreover, MRP4 was identified as the main contributor based on chemical inhibition and gene silencing approaches. CONCLUSIONS: CYP1A1 and CYP2C9, UGT1A1, UGT1A7, UGT1A8 and UGT1A9, and MRP4 all played important roles in the metabolism and disposition of bavachin.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Flavonoides/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Microssomos Hepáticos/enzimologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Cães , Glucuronosiltransferase/genética , Haplorrinos , Células HeLa , Humanos , Isoenzimas , Cinética , Desintoxicação Metabólica Fase I , Desintoxicação Metabólica Fase II , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Coelhos , Ratos , Especificidade por Substrato , Suínos , Porco MiniaturaRESUMO
Bu-Zhong-Yi-Qi-Tang (BZYQT), a famous traditional Chinese medicine prescription (TCMP), has been extensively used for conditioning sub-health status and diseases caused by spleen-qi deficiency in China for over 700 years. BZYQT is prevalent not only in China, but also in Japan and South Korea for the clinical treatment of chronic diseases, such as fatigue, tuberculosis and loss of appetite after surgery. However, due to a lack of research on the holistic metabolism of BZYQT, the in vivo bioactive components of BZYQT remain unclear, hindering further study of its in vivo mechanism of action and quality control. In the present study, a four-step integrated strategy based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS) was established to systematically screen the in vivo xenobiotics of BZYQT. Ultimately, a total of 162 xenobiotics (59 prototypes and 103 metabolites) were identified or tentatively characterized, including 48 in plasma, 147 in urine and 58 in feces, while the in vivo metabolic profile of atractylenolide III (a major component of BZYQT) was elucidated for the first time. The xenobiotics of BZYQT mainly included flavonoids from Astragali Radix, Glycyrrhizae Radix et Rhizoma and Citrus reticulatae Pericarpium; lactones from Angelicae Sinensis Radix and Atractylodis Macrocephalae Rhizoma; and triterpenoid saponins from Cimicifugae Rhizoma. After oral administration, BZYQT-related components underwent diverse metabolic pathways. Among them, flavonoids mainly underwent glucuronidation, sulfation and demethylation, while lactones mainly underwent hydroxylation and acetylcysteine conjugation, and deglycosylation was the major metabolic reaction of saponins. Our investigation gives a comprehensive analysis of the metabolic characteristics of BZYQT and will provide an important basis for further studying the pharmacokinetics of BZYQT to explore its in vivo disposal features and discover its in vivo bioactive components.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/farmacocinética , Flavonoides/análise , Espectrometria de Massas em Tandem/métodos , Triterpenos/análise , Administração Oral , Animais , Fezes/química , Flavonoides/sangue , Flavonoides/urina , Lactonas/metabolismo , Masculino , Desintoxicação Metabólica Fase I , Desintoxicação Metabólica Fase II , Metaboloma , Estrutura Molecular , Ratos Sprague-Dawley , Sesquiterpenos/metabolismo , Triterpenos/sangue , Triterpenos/urinaRESUMO
ETHNOPHARMACOLOGICAL EVIDENCE: With fast development and high pace life in modern society, autoimmune diseases like inflammatory bowel disease had become increasingly common. Bu-Zhong-Yi-Qi-Tang (BZYQT), a famous traditional Chinese medicine prescription (TCMP), has been used for 700 years mainly in Eastern Asia countries for the treatment of gastrointestinal and respiratory disorder, and weakness after serious diseases. These diseases were proved to be closely related to human immune system, among which, mucosal immune system is the largest immune system. So it is necessary to discover the mucosal immune related bioactive components of BZYQT. AIM OF THE STUDY: To evaluate the mucosal immunomodulatory bioactivity of BZYQT and ingredients. MATERIALS AND METHODS: Peyer's patches were collected from mice administrated orally with BZYQT, its related Octadecylsilane (ODS) fractions and polysaccharide part. Productions of several cytokines including IL-2, IL-4, IL-5, and IFN-γ from T lymphocytes were tested with enzyme linked immunosorbent assay (ELISA) by Peyer's patch cells ex vivo experiments. Chemical profile including low molecular part and polysaccharide part were investigated. Low molecular part of BZYQT and related ODS fractions were analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) based on LC-MS information from self-established compound library. exclusion chromatography, and chemical property has been analyzed. RESULT: Three-days' administration of BZYQT enhanced productions of IL-4 and IFN-γ in T lymphocytes of Peyer's patches in addition to IL-2. Some hydrophobic low molecular weight fractions (30%, 70% and 100% MeOH ODS fraction), which were fractionated from BZYQT by ODS column chromatography, showed enhancing or suppressive effects on productions of IL-2, IL-4 or IL-5 in T lymphocytes of Peyer's patches after oral administration. Besides, 161 components from hydrophobic low molecular weight fractions of BZYQT were unequivocally identified or tentatively characterized by UPLC-Q/TOF-MS according to retention time behaviors and fragments, and most of them were flavonoids and saponins from Glycyrrhizae Radix, Citri Reticulatae Pericarpium, and Cimicifugae Rhizoma. Polysaccharide part was separated and purified both by anion-exchange and size. BZYQT also contained at least one neutral and three weakly or strongly acidic polysaccharides, and analysis of their chemical properties indicated that a neutral polysaccharide was glucan, and acidic polysaccharides possessed heteroglycan and pectic arabinogalactan features. Murine administration of polysaccharide fractions of BZYQT induced different changes on functions of T lymphocytes in Peyer's patches from hydrophobic low molecular weight fractions. By experiment using intranasally-immunized mice, BZYQT negatively regulated antibody response in lung as combinatorial actions of its low molecular weight ingredients and polysaccharides. CONCLUSION: BZYQT contains several low and macromolecular weight ingredients, which affect to immune-function of T lymphocytes in Peyer's patches, and the formula expresses its regulative activity on lower respiratory immune system through combinatorial actions of these ingredients on immunocompetent cells in Peyer's patches.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Fatores Imunológicos/farmacologia , Animais , Citocinas/imunologia , Medicamentos de Ervas Chinesas/química , Feminino , Imunidade nas Mucosas , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Fatores Imunológicos/química , Vacinas contra Influenza/administração & dosagem , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Medicina Tradicional Chinesa , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Nódulos Linfáticos Agregados/efeitos dos fármacos , Nódulos Linfáticos Agregados/imunologia , Polissacarídeos/farmacologia , Silanos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Neobavaisoflavone (NBIF), a phenolic compound isolated from Psoralea corylifolia L., possesses several significant biological properties. However, the pharmacokinetic behaviors of NBIF have been characterized as rapid oral absorption, high clearance, and poor oral bioavailability. We found that NBIF underwent massive glucuronidation and oxidation by human liver microsomes (HLM) in this study with the intrinsic clearance (CLint) values of 12.43, 10.04, 2.01, and 6.99⯵L/min/mg for M2, M3, M4, and M5, respectively. Additionally, the CLint values of G1 and G2 by HLM were 271.90 and 651.38⯵L/min/mg, respectively, whereas their respective parameters were 59.96 and 949.01⯵L/min/mg by human intestine microsomes (HIM). Reaction phenotyping results indicated that CYP1A1, 1A2, 2C8, and 2C19 were the main contributors to M4 (34.96⯵L/min/mg), M3 (29.45⯵L/min/mg), M3 (13.16⯵L/min/mg), and M2 (63.42⯵L/min/mg), respectively. UGT1A1, 1A7, 1A8, and 1A9 mainly catalyzed the formation of G1 (250.87⯵L/min/mg), G2 (438.15⯵L/min/mg), G1 (92.68⯵L/min/mg), and G2 (1073.25⯵L/min/mg), respectively. Activity correlation analysis assays showed that phenacetin-N-deacetylation was strongly correlated to M3 (râ¯=â¯0.860, pâ¯=â¯0.003) and M4 (râ¯=â¯0.775, pâ¯=â¯0.014) in nine individual HLMs, while significant activity correlations were detected between paclitaxel-6-hydroxylation and M2 (râ¯=â¯0.675, pâ¯=â¯0.046) and M3 (râ¯=â¯0.829, pâ¯=â¯0.006). There was a strong correlation between ß-estradiol-3-O-glucuronide and G1 (râ¯=â¯0.822, pâ¯=â¯0.007) and G2 (râ¯=â¯0.689, pâ¯=â¯0.040), as well as between propofol-O-glucuronidation and G1 (râ¯=â¯0.768, pâ¯=â¯0.016) and G2 (râ¯=â¯0.860, pâ¯=â¯0.003). Moreover, the phase I metabolism and glucuronidation of NBIF revealed marked species differences, and mice are the best animal model for investigating the metabolism of NBIF in humans. Taken together, characterization of NBIF-related metabolic pathways involving in CYP1A1, 1A2, 2C8, 2C19, and UGT1A1, 1A7, 1A8, 1A9 are helpful for understanding the pharmacokinetic behaviors and conducting in-depth pharmacological studies.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Isoflavonas/farmacocinética , Microssomos Hepáticos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Ensaios Enzimáticos , Glucuronídeos/metabolismo , Haplorrinos , Humanos , Hidroxilação , Mucosa Intestinal/metabolismo , Intestinos/citologia , Fígado/citologia , Fígado/metabolismo , Camundongos , Oxirredução , Coelhos , Ratos , Especificidade da Espécie , Espectrometria de Massas em TandemRESUMO
Cycloicaritin is a bioactive natural phenolic compound from Epimedium species. However, the glucuronidation and excretion which would influence oral bioavailability and pharmacokinetics of cycloicaritin still remain unknown. Here we aimed to establish UGT1A1 stably transfected HeLa cells, and to determine the contributions of BCRP and MRPs transporters to excretion of cycloicaritin-3-O-glucuronide. First, ß-estradiol was used to validate the expression of active UGT1A1 protein in engineered HeLa1A1 cells. Furthermore, Ko143 (5 and 20⯵M) led to a significant decrease (42.4%-63.8%, pâ¯<â¯0.01) in CICT-3-G excretion and obvious accumulation (19.7%-54.2%, pâ¯<â¯0.05) of intracellular CICT-3-G, while MK571 (5 and 20⯵M) caused a significant reduction (46.8%-64.8%, pâ¯<â¯0.05) in the excretion and obvious elevation (50.7%-85.2%, pâ¯<â¯0.01) of intracellular level of CICT-3-G. Furthermore, BCRP knocked-down brought marked reduction in excretion rates of CICT-3-G (26.0%-42.2%, pâ¯<â¯0.01), whereas MRP1 and MRP4-mediated silencing led to significant decrease in the excretion of CICT-3-G (23.8%-35.4%, pâ¯<â¯0.05 for MRP1 and 11.9%-16.0%, pâ¯<â¯0.05 for MRP4). By contrast, neither CICT-3-G excretion nor CICT-3-G accumulation altered in MRP3 knocked-down cells as compared to scramble cells. Taken together, BCRP, MRP1 and MRP4 were identified as the most important contributors for CICT-3-G excretion. Meanwhile, the UGT1A1 modified HeLa cells were a simple and practical tool to study UGT1A1-mediated glucuronidation and to characterize BCRP and MRPs-mediated glucuronide transport at a cellular level.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Cromatografia Líquida de Alta Pressão , Glucuronídeos/química , Glucuronosiltransferase/biossíntese , Células HeLa , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas de Neoplasias/químicaRESUMO
BACKGROUND AND PURPOSE: The clinical use of arsenic trioxide (As2O3) for treating acute promyelocytic leukemia (APL) is limited due to its severe cardiotoxicity. The possible mechanisms of As2O3-induced cardiotoxicity include DNA fragmentation, reactive oxygen species (ROS) generation, cardiac ion channel changes and apoptosis. The present study is designed to investigate the protective effects of imperatorin and sec-O-glucosylhamaudol and to explore their mechanistic involvement in As2O3-induced cytotoxicity. EXPERIMENTAL METHODS: Cell viability assay, Lactate dehydrogenase (LDH) release, Acridine orange/ethidium bromide (AO/EB) double staining, Caspase-3 activity assay, ROS generation, cellular calcium levels, mRNA expression levels by qRT-PCR and protein expression levels by Western blotting were measured in H9c2 cells in combination with As2O3 and imperatorin or sec-O-glucosylhamaudol. KEY RESULTS: We observed that H9c2 cells treated with imperatorin or sec-O-glucosylhamaudol were more resistant to As2O3-induced cell death. Both imperatorin and sec-O-glucosylhamaudol reduced H9c2 cell apoptosis, but both imperatorin and sec-O-glucosylhamaudol had no effects on Caspase-3 activity and intracellular calcium accumulation. Furthermore, imperatorin was capable of suppressing ROS generation, while sec-O-glucosylhamaudol did not show this effect. Moreover, imperatorin and sec-O-glucosylhamaudol triggered Nrf2 activation, which resulted in upregulation of downstream phase II metabolic enzymes and antioxidant protein/enzyme, probably offering cellular protection to As2O3-induced cardiotoxicity via the Nrf2 signal pathway. CONCLUSIONS AND IMPLICATIONS: Imperatorin and sec-O-glucosylhamaudol can ameliorate As2O3-induced cytotoxicity and apoptosis in H9c2 cells, the mechanisms probably related to antioxidation. As2O3 in combination with imperatorin or sec-O-glucosylhamaudol could be considered as a novel strategy to expand the clinical application of As2O3.