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1.
Anal Chem ; 96(31): 12927-12935, 2024 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-39041225

RESUMO

Mas-related G protein-coupled receptor X2 (MrgprX2) plays a crucial role in anaphylactoid reactions and allergic diseases. Some antagonists with reasonable potency and selectivity have been reported. Cell membrane chromatography (CMC) is effective for discovering ligands. Protein-tag-based CMC models (e.g., SNAP tags and HALO tags) have enhanced performance but also increased nonspecific adsorption of small molecules. The Avi tag, a short peptide sequence, binds biotin specifically via BirA catalysis. Our study showed that 2-iminobiotin (IB) can be a BirA substrate, enabling the development of a new cell membrane stationary phase (CMSP) based on the chemical properties (modifying carboxyl silica gel and specifically labeling the Avi tag) of IB. First, we constructed the MrgprX2-Avi-tag HEK293T cell line. Next, we synthesized IB-modified silica gel (SiO2-IB) stepwise. Finally, we immobilized Avi-tagged MrgprX2 cell membranes on SiO2-IB under BirA catalysis. We characterized the developed CMSP and used it to establish a MrgprX2-Avi-tag/CMC-HPLC/MS two-dimensional screening platform, successfully screening vitexicarpin fromViticis Fructus extract via a 2D/CMC platform. In vitro and in vivo experiments confirmed that vitexicarpin targets the MrgprX2 receptor, demonstrating antiallergic effects. Our IB-Avi tag-based CMC approach effectively decreased nonspecific adsorption of the screening materials. The Avi-tag-based 2D/CMC platform is suitable for screening potential drug candidates.


Assuntos
Membrana Celular , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Células HEK293 , Membrana Celular/metabolismo , Animais , Receptores de Neuropeptídeos/metabolismo , Receptores de Neuropeptídeos/antagonistas & inibidores , Camundongos , Cromatografia Líquida de Alta Pressão , Proteínas do Tecido Nervoso
2.
Anal Chem ; 96(26): 10851-10859, 2024 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-38912707

RESUMO

Mitochondrial Membrane Chromatography (MMC) is a bioaffinity chromatography technique developed to study the interaction between target proteins embedded in the mitochondrial membrane and their ligand compounds. However, the MMC stationary phases (MMSP) prepared by chemical immobilization are prone to nonspecific binding in candidate agent screening inevitably. To address these challenges, Twin Strep-Tag/Strep Tactin was employed to establish a specific affinity system in the present study. We prepared a carnitine palmitoyltransferase 1A (CPT1A) MMSP by specifically linking Strep-tactin-modified silica gel with the Twin Strep-Tag on the CPT1A-oriented mitochondrial membrane. This Twin Strep-Tag/Strep Tactin modified CPT1A/MMC method exhibited remarkably better retention behavior, longer stationary phase lifespan, and higher screening specificity compared with previous MMC systems with glutaraldehyde immobilization. We adopted the CPT1A-specific MMC system in screening CPT1A ligands from traditional Chinese medicines, and successfully identified novel candidate ligands: ononin, isoliquiritigenin, and aloe-emodin, from Glycyrrhiza uralensis Fisch and Senna tora (L.) Roxb extracts. Biological assessments illustrated that the compounds screened promote CPT1A enzyme activity without affecting CPT1A protein expression, as well as effectively reduce the lipid droplets and triglyceride levels in the high fat induction HepG2 cells. The results suggest that we have developed an MMC system, which is promising for studying the bioaffinity of mitochondrial membrane proteins to candidate compounds. This system provides a platform for a key step in mitochondrial medicine discovery, especially for bioactive molecule screening from complex herbal extracts.


Assuntos
Carnitina O-Palmitoiltransferase , Metabolismo dos Lipídeos , Membranas Mitocondriais , Humanos , Carnitina O-Palmitoiltransferase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Cromatografia de Afinidade , Ligantes
3.
Anal Bioanal Chem ; 415(7): 1371-1383, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36651973

RESUMO

In this study, a novel cell membrane chromatography (CMC) model was developed to investigate cluster of differentiation 147 (CD147) targeted anti-tumor drug leads for specific screening and ligand-receptor interaction analysis by SNAP-tagged CD147 fusion protein conjugation and polystyrene microspheres (PS) modification. Traditional Chinese medicines (TCMs) are widely used in the treatment of cancer. CD147 plays important roles in tumor progression and acts as an attractive target for therapeutic intervention; therapeutic drugs for CD147-related cancers are limited to date. Thus, a screening method for active components in TCMs is crucial for the further research and development of CD147 antagonists. However, improvement is still needed to perform specific and accurate drug lead screening using the CMC-based method. Recently, our group developed a covalently immobilized receptor-SNAP-tag/CMC model using silica gel as carrier. Besides the carboxyl group on multi-step modified silica particles, the amino group of benzyl-guanine (BG, substrate of SNAP-tag) also possesses reactivity towards the carboxyl group on available carboxyl-modified PS. Herein, we used PS as carrier and an extended SNAP-tag with CD147 receptor to construct the PS-BG-CD147/CMC model for active compound investigation coupled with HPLC/MS and applied this coupled PS-BG-CD147/CMC-HPLC/MS two-dimensional system to drug lead screening from Nelumbinis Plumula extract (NPE) sample. In addition, to comprehensively verify the pharmacological effects of screened ingredients, a cell proliferation inhibition assay was performed, and the interaction between the ingredients and CD147 was studied by the frontal analysis method. This study developed a high-throughput PS-based CMC screening platform, which could be widely applied and utilized in chromatographic separation and drug lead discovery.


Assuntos
Medicamentos de Ervas Chinesas , Medicamentos de Ervas Chinesas/química , Poliestirenos/análise , Microesferas , Cromatografia Líquida de Alta Pressão/métodos , Membrana Celular/química
4.
Immunopharmacol Immunotoxicol ; 45(6): 672-681, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37339357

RESUMO

BACKGROUND: Mast cells (MCs) are important effector cells in anaphylaxis and anaphylactic disease. 3',4',5,7-tetrahydroxyflavone (THF) presents in many medicinal plants and exerts a variety of pharmacological effects. In this study, we evaluated the effect of THF on C48/80-induced anaphylaxis and the mechanisms underlying its effects, including the role of secreted phosphoprotein 1 (SPP1), which has not been reported to IgE-independent MC activation. RESULTS: THF inhibited C48/80-induced Ca2+ flow and degranulation via the PLCγ/PKC/IP3 pathway in vitro. RNA-seq showed that THF inhibited the expression of SPP1 and downstream molecules. SPP1 is involved in pseudo-anaphylaxis reactions. Silencing SPP1 affects the phosphorylation of AKT and P38. THF suppressed C48/80-induced paw edema, hypothermia and serum histamine, and chemokines release in vivo. CONCLUSIONS: Our results validated SPP1 is involved in IgE-independent MC activation anaphylactoid reactions. THF inhibited C48/80-mediated anaphylactoid reactions both in vivo and in vitro, suppressed calcium mobilization and inhibited SPP1-related pathways.


Assuntos
Anafilaxia , Humanos , Anafilaxia/induzido quimicamente , Anafilaxia/tratamento farmacológico , Luteolina/farmacologia , Osteopontina/metabolismo , Osteopontina/farmacologia , Mastócitos , Inflamação/metabolismo , Degranulação Celular , Imunoglobulina E/metabolismo
5.
Clin Exp Allergy ; 52(1): 46-58, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-33999474

RESUMO

BACKGROUND: Allergic asthma is a common inflammatory lung disease and a major health problem worldwide. Mast cells (MCs) play a key role in the early-stage pathophysiology of allergic asthma. Substance P (SP) functions in neurogenic inflammation by activating MCs, and therefore, it may to participate in the occurrence and development of asthma. OBJECTIVE: We examined the relationship between SP and lung inflammation, and also whether SP can directly trigger asthma. METHODS: We measured the number of peripheral blood eosinophils, neutrophils and basophils and evaluated the levels of IgE and SP in blood samples of 86 individuals with allergic asthma. Serum IgE and SP levels were also determined in 29 healthy individuals. C57BL/6 mice were subjected to different doses of SP, and bronchoalveolar lavage fluid (BALF) was collected to count the inflammatory cells. Lung tissues were analysed using histopathological methods to evaluate lung peribronchial inflammation, fibrosis and glycogen deposition. Levels of IgE, interleukin (IL)-1, IL-2, IL-4, IL-5, IL-13, IL-17 and IFN-γ were determined in mouse serum. RESULTS: Substance P levels were increased in the serum samples of patients with asthma. SP induced mouse lung peribronchial inflammation, fibrosis and glycogen deposition, with high levels of Th2-related cytokines such as IL-4, IL-5 and IL-13 observed in the BALF. Furthermore, low level of total IgE was noted in the serum, and SP had little effect on MC-deficient kitW-sh/W-sh mice. CONCLUSIONS & CLINICAL RELEVANCE: Substance P levels increased significantly in serum of asthmatic patients and independently associated with the risk of asthma. Furthermore, SP induced Th2 lung inflammation in mice, which was dependent on MCs.


Assuntos
Pneumonia , Substância P , Animais , Líquido da Lavagem Broncoalveolar , Citocinas , Modelos Animais de Doenças , Humanos , Pulmão , Mastócitos , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/patologia
6.
Anal Bioanal Chem ; 414(19): 5741-5753, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35697810

RESUMO

Pseudo-allergic reactions (PARs) are IgE-independent hypersensitivity reactions. Mas-related G protein-coupled receptor-X2 (MrgX2) was proved the key receptor of PAR. The anti-pseudo-allergic compound discovery based on MrgX2 was of great value. Cell membrane chromatography (CMC) based on MrgX2 provides a convenient and effective tool in anti-pseudo-allergic compound screening and discovery, and further improvements of this method are still needed. In this work, SNAP-tag was introduced at C-terminal of Mas-related G protein-coupled receptor (MrgX2-SNAP-tag), and an MrgX2-SNAP-tag/CMC model was then conducted using CMC technique. Comparative experiments showed that the new model not only satisfied the good selectivity and specificity of screening but also exhibited more stable and longer life span than traditional MrgX2/CMC model. By coupling with HPLC-MS, two compounds were screened out from Arnebiae Radix and identified as shikonin and acetylshikonin. Nonlinear chromatography was performed to study the interactions between two screened compounds and MrgX2, and binding constant (KA) of shikonin and acetylshikonin with MrgX2 were 2075.67 ± 0.34 M-1 and 32201.36 ± 0.35 M-1, respectively. Furthermore, ß-hexosaminidase and histamine release assay in vitro demonstrated that shikonin (1-5 µM) and acetylshikonin (2.5-10 µM) could both antagonize C48/80-induced allergic reaction. In conclusion, the MrgX2-SNAP-tag/CMC could be a reliable model for screening pseudo-allergy-related components from complex systems.


Assuntos
Antialérgicos , Receptores de Neuropeptídeos , Antialérgicos/análise , Antialérgicos/metabolismo , Antialérgicos/farmacologia , Membrana Celular/metabolismo , Cromatografia Líquida , Espectrometria de Massas , Mastócitos/química , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/análise , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/metabolismo
7.
J Sep Sci ; 45(14): 2498-2507, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35561141

RESUMO

Cell membrane chromatography is an effective method for screening bioactive components acting on specific receptors in complex systems, which maintains the biological activity of the membrane receptors and improves screening efficiency. However, traditional cell membrane chromatography suffers from poor stability, resulting in a limited life span and low reproducibility, greatly limiting the application of this method. To address this problem, cyanuric chloride-decorated silica gel was used for the covalent immobilization of the cell membranes. Cyanuric chloride reacts with amino groups on the cell membranes and membrane receptors to form covalent bonds. In this way, the cell membranes are not easy to fall off. The column life of the cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography column was extended to more than 8 days, whereas the column life of the normal cell membrane chromatography column dropped sharply in the first 3 days. A cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography online HPLC-IT-TOF-MSn system was applied for screening drug leads from Trifolium pratense L. One potential drug lead, formononetin, which acts on the epidermal growth factor receptor, was screened. Our strategy of covalently immobilizing cell membrane receptors also improved the stability of cell membrane chromatography.


Assuntos
Medicamentos de Ervas Chinesas , Receptores ErbB , Membrana Celular/química , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/análise , Receptores ErbB/metabolismo , Reprodutibilidade dos Testes
8.
Anal Chem ; 93(36): 12296-12304, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34347443

RESUMO

Scanning ion conductance microscopy (SICM) is a type of in situ measurement technology for noncontact detection of samples in electrolytes with nanoscale resolution and has been used increasingly in biomedical and electrochemical fields in recent years. However, there is an inherent contradiction in the technique that makes SICM's sensitivity and accuracy difficult to balance. Higher sensitivity allows for faster probe speeds and higher scanning reliability but leads to lower accuracy, and vice versa. To resolve this problem, an adaptive sensitivity scanning method is proposed here that is designed to increase SICM's imaging efficiency without reducing its scanning reliability and accuracy. In the proposed scanning method, the sensitivity is automatically switched via the bias voltage based on the probe-sample distance. When the probe is located far away from the sample, the probe then predetects the sample position rapidly with high sensitivity. When the sample has been sensed in the high-sensitivity phase, the probe then detects the sample with low sensitivity. The basic theory and the feasibility of the alterable sensitivity detection strategy is also studied using the finite element method (FEM) and by performing experiments in this work. Finally, through testing of the standard silicon and polydimethylsiloxane (PDMS) samples, the proposed method is shown to increase SICM imaging efficiency significantly by up to 5 times relative to the conventional hopping mode without sacrificing the scanning accuracy and reliability.


Assuntos
Microscopia , Íons , Cintilografia , Reprodutibilidade dos Testes
9.
J Med Virol ; 93(5): 3143-3151, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33580518

RESUMO

Since December 2019, the new coronavirus (also known as severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2, 2019-nCoV])-induced disease, COVID-19, has spread rapidly worldwide. Studies have reported that the traditional Chinese medicine Salvia miltiorrhiza possesses remarkable antiviral properties; however, the anti-coronaviral activity of its main components, salvianolic acid A (SAA), salvianolic acid B (SAB), and salvianolic acid C (SAC) is still debated. In this study, we used Cell Counting Kit-8 staining and flow cytometry to evaluate the toxicity of SAA, SAB, and SAC on ACE2 (angiotensin-converting enzyme 2) high-expressing HEK293T cells (ACE2h cells). We found that SAA, SAB, and SAC had a minor effect on the viability of ACE2h cells at concentrations below 100 µM. We further evaluated the binding capacity of SAA, SAB, and SAC to ACE2 and the spike protein of 2019-nCoV using molecular docking and surface plasmon resonance. They could bind to the receptor-binding domain (RBD) of the 2019-nCoV with a binding constant (KD ) of (3.82 ± 0.43) e-6 M, (5.15 ± 0.64)e-7 M, and (2.19 ± 0.14)e-6 M; and bind to ACE2 with KD (4.08 ± 0.61)e-7 M, (2.95 ± 0.78)e-7 M, and (7.32 ± 0.42)e-7 M, respectively. As a result, SAA, SAB, and SAC were determined to inhibit the entry of 2019-nCoV Spike pseudovirus with an EC50 of 11.31, 6.22, and 10.14 µM on ACE2h cells, respectively. In conclusion, our study revealed that three Salvianolic acids can inhibit the entry of 2019-nCoV spike pseudovirus into ACE2h cells by binding to the RBD of the 2019-nCoV spike protein and ACE2 protein.


Assuntos
Alcenos/farmacologia , Enzima de Conversão de Angiotensina 2/metabolismo , Benzofuranos/farmacologia , Ácidos Cafeicos/farmacologia , Lactatos/farmacologia , Polifenóis/farmacologia , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Alcenos/química , Enzima de Conversão de Angiotensina 2/química , Benzofuranos/química , Ácidos Cafeicos/química , Sobrevivência Celular , Células HEK293 , Humanos , Lactatos/química , Estrutura Molecular , Polifenóis/química , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus , Tratamento Farmacológico da COVID-19
10.
Anal Biochem ; 633: 114392, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34597615

RESUMO

Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis. Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions. For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively. We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH2 groups for covalent immobilization of the capture antibody. Through amide condensation, a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively. This strategy improved the quantity, activity and stability of the immobilized antibody. We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood. Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA, while offering similar sensitivity, specificity, and accuracy. Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment.


Assuntos
Anafilaxia/sangue , Ensaio de Imunoadsorção Enzimática , Proteínas do Tecido Nervoso/sangue , Papel , Receptores Acoplados a Proteínas G/sangue , Receptores de Neuropeptídeos/sangue , Anafilaxia/imunologia , Humanos , Proteínas do Tecido Nervoso/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores de Neuropeptídeos/imunologia
11.
J Immunol ; 203(7): 1701-1714, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31484729

RESUMO

Intrathecal morphine infusion is often applied to treat chronic pain related to cancer and other conditions. However, persistent pain can be caused by nerve compression because of granuloma formation. In this study, a mouse model of morphine-induced granuloma formation by intrathecal catheterization morphine infusion into the atlanto-occipital membrane of the foramen magnum was established in wild-type mice, MrgprB2 mutant (MrgprB2-/-) mice, and in mast cell-deficient W-sash c-kit mutant (KitW-sh/W-sh) mice. Heat-related pain after surgery was performed to investigate the antipain effect of morphine. H&E staining and immunofluorescence staining of the spinal cord were applied to analyze the mechanism of granuloma formation. Morphine-induced mast cell degranulation was assessed by measuring the Ca2+ influx and mediator release. Anaphylactoid reactions were measured after s.c. morphine infusion to the paws. Chemokine release by mast cells was determined by Human XL Cytokine Array. Experiments with wild-type, MrgprB2 mutant, and mast cell-deficient W-sash c-kit mutant mice demonstrated that morphine activated mast cells and inflammatory cell aggregation through MrgprB2 in intrathecal infusion sites. The chemokine production of human mast cells demonstrated that granuloma formation is correlated with chemokines release. In addition, morphine activated mouse primary mast cells and de novo chemokine synthesis via the MRGPRX2 in human LAD2 cells. We concluded that granuloma formation during intrathecal morphine infusion was associated with MrgprB2/X2. Reducing MRGPRX2 potentially blocks morphine-induced side effects, including granuloma formation.


Assuntos
Granuloma/imunologia , Mastócitos/imunologia , Morfina/efeitos adversos , Dor/imunologia , Receptores Acoplados a Proteínas G/imunologia , Medula Espinal/imunologia , Animais , Quimiocinas/genética , Quimiocinas/imunologia , Forame Magno/imunologia , Forame Magno/patologia , Granuloma/patologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Injeções Espinhais , Masculino , Mastócitos/patologia , Camundongos , Camundongos Knockout , Morfina/farmacologia , Dor/tratamento farmacológico , Dor/patologia , Receptores Acoplados a Proteínas G/genética , Medula Espinal/patologia
12.
Anal Bioanal Chem ; 413(11): 2995-3004, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33608752

RESUMO

Traditional Chinese medicines played an important role in the treatment of COVID-19 in 2020. Ephedra sinica, one of the major constituent herbs of multi-component herbal formula, has been widely used to treat COVID-19 in China. However, its active components are still unclear. The objectives of this study are to screen and evaluate active components from the traditional Chinese medicine Ephedra sinica for the treatment of COVID-19. In our study, we established an ACE2/CMC bioaffinity chromatography model, and then developed an ACE2/CMC-HPLC-IT-TOF-MS system for the active compounds screening and identification from Ephedra sinica extract. We performed molecular docking and surface plasmon resonance (SPR) assays to assess the binding characteristics (binding mode and KD value). We used CCK-8 staining to assess the toxicity of screened compounds, and also used SARS-CoV-2 pseudovirus to observe the viropexis effect of screened compounds in ACE2h cells. In this current work, one fraction was fished out, separated and identified as ephedrine (EP), pseudoephedrine (PEP), and methylephedrine (MEP). Binding assays showed that the three compounds could bind with ACE2 in a special way to some amino acid residues, similar to the way SARS-CoV-2 bound with ACE2. Additionally, the three compounds, especially EP, can inhibit the entrance of SARS-CoV-2 spike pseudovirus into ACE2h cells because they can reduce the entrance ratio of pseudovirus in the pseudovirus model. Overall, the ACE2/CMC-HPLC-IT-TOF-MS system was established and verified to be suitable for ACE2-targeted bioactive compound screening. EP, PEP, and MEP with ACE2-binding features were screened out from Ephedra sinica, and acted as blockers inhibiting SARS-CoV-2 spike pseudovirus entering ACE2h cells.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Medicamentos de Ervas Chinesas/farmacologia , Ephedra sinica , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , Antivirais/isolamento & purificação , COVID-19/metabolismo , China , Cromatografia Líquida de Alta Pressão , Descoberta de Drogas , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Ephedra sinica/química , Efedrina/análogos & derivados , Efedrina/isolamento & purificação , Efedrina/farmacologia , Células HEK293 , Humanos , Espectrometria de Massas , Simulação de Acoplamento Molecular , SARS-CoV-2/fisiologia , Internalização do Vírus/efeitos dos fármacos
13.
Phytother Res ; 35(6): 3181-3193, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33893660

RESUMO

Anaphylactoid reactions are potentially fatal allergic diseases caused by mast cells (MCs), which release histamine and lipid mediators under certain stimuli. Therefore, there is an urgent need to develop new drug candidates to treat anaphylactoid reactions. The MrgX2 receptor mediates anaphylactoid reactions that cause inflammatory diseases. Cortex dictamni is a Chinese herb used for treating allergy-related diseases; however, its active compound is still unknown and its mechanism of action has not yet been reported. The aim of this study was to screen the anti-anaphylactoid compound from C. dictamni extracts. An MrgX2/CMC-HPLC method was established for screening MrgX2-specific compounds retained from the alcohol extract of C. dictamni. A mouse model of hindpaw extravasation was used to evaluate the anti-anaphylactoid effect of this ingredient. Intracellular Ca2+ mobilization was assessed using a calcium imaging assay. Enzyme immunoassays were performed to measure cytokine and chemokine release levels. The molecular signaling pathways were explored by western blotting. As a result, dictamnine was identified as an effective compound using the MrgX2/CMC method, which remarkably suppressed MC intracellular Ca2+ mobilization and the release of de novo degranulated substances, and inhibited PKC-PLCγ-IP3R-associated protein signaling molecules. Hence, dictamnine is a novel therapeutic candidate for anaphylactoid reactions via MrgX2.


Assuntos
Anafilaxia/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Animais , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Histamina/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais
14.
Toxicol Appl Pharmacol ; 392: 114921, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32061592

RESUMO

Angioedema may occur during local anesthetic (LA) injection in the perioperative period. Histaminergic angioedema is the most common form of angioedema. It has been reported that LA is a potential exogenous ligand for histamine receptor 1 (H1R). Whether H1R participates in LA-induced angioedema is still controversial. By using a constructed H1R high-expressed cell model, siRNA transfection, pharmacologic means, and genetically modified animal models, here we showed that H1R mediated LA-induced hyperpermeability. LA with uncycled N-methyl scaffold in the side chain (procaine, tetracaine and lidocaine) had a better strength of drug-H1R affinity than that for LA with cycled N atom (bupivacaine and ropivacaine) by the molecular docking assay and equilibrium dissociation constant (KD values) obtained from the cell membrane chromatography (CMC) relative standard method. Procaine, tetracaine, and lidocaine triggered big calcium mobilization in H1R-HEK293 cells and human umbilical vein endothelial cells (HUVECs) but much weaker in NC-HEK293 cells or H1R knockdown HUVECs. Besides, the results of transendothelial resistance measurement, paracellular flux assay and immunofluorescence showed that procaine induced H1R-dependent hyperpermeability, which involved in PLCγ/IP3R/PKC, ERK1/2, Akt signaling pathways, downstream vascular endothelial cadherin (VE-cad) destabilization. Furthermore, H1R gene knockout prevented paw swelling and vascular leakage caused by procaine, tetracaine, and lidocaine in vivo. This study supported a key role of H1R in LA-induced angioedema, and suggested that in the design of LA structure, the ring formation of the N-methyl scaffold on the side chain can properly avoid the angioedema.


Assuntos
Anestésicos Locais/farmacologia , Angioedema/patologia , Permeabilidade Capilar/efeitos dos fármacos , Receptores Histamínicos H1/metabolismo , Animais , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Mastócitos , Camundongos , Camundongos Knockout , Distribuição Aleatória
15.
Contact Dermatitis ; 81(3): 184-193, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31006867

RESUMO

BACKGROUND: Retinoic acid (RA)-induced dermatitis is the most frequent side-effect limiting its widespread use. However, the exact mechanisms triggering dermatitis are not fully understood, including the role of skin mast cells. The newly discovered Mas-related G-protein-coupled receptor-X2 (MRGPRX2) in mast cells mediates pseudoallergic drug reactions in several types of dermatitis. A possible contribution of MRGPRX2 to contact dermatitis induced by RA has hitherto not been examined. OBJECTIVES: To investigate whether all-trans-RA (ATRA) activates mast cells via MRGPRX2/MrgprB2 (the mouse orthologue), contributing to the pathogenesis of retinoid-induced dermatitis. METHODS: Wild-type (WT) and MrgprB2-/- mice were treated with topical ATRA to observe local inflammation and mast cell degranulation in vivo by the use of haematoxylin and eosin and immunofluorescence staining. Release of histamine and release of ß-hexosaminidase were measured and calcium influx was detected in Laboratory of Allergic Disease 2 (LAD2) cells with specific knockdown targeting MRGPRX2 by small interfering RNA (siRNA) and in primary cells from MrgprB2-/- mice. RESULTS: As compared with WT mice, MrgprB2-/- mice showed resistance to ATRA-triggered contact dermatitis and local inflammatory reactions in the paws. ATRA activated mast cells via the MrgprB2 pathway in murine cells, and via the MRGPRX2 pathway in human mast cells. CONCLUSIONS: ATRA-induced dermatitis could be achieved by activating mast cells via MRGPRX2/MrgprB2, which may provide a potential therapy target to reduce the side-effect.


Assuntos
Degranulação Celular/efeitos dos fármacos , Dermatite de Contato/etiologia , Mastócitos/fisiologia , Receptores Acoplados a Proteínas G/genética , Tretinoína/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular , Dermatite , Dermatite de Contato/genética , Técnicas de Silenciamento de Genes , Histamina/metabolismo , Humanos , Masculino , Mastócitos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Tretinoína/toxicidade , beta-N-Acetil-Hexosaminidases/metabolismo
16.
Environ Monit Assess ; 191(3): 171, 2019 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783770

RESUMO

Little is known about the extent of heavy metal accumulation in traditional Chinese medicines (TCMs). In this study, the levels of lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg) in traditional animal medicines were monitored using inductively coupled plasma mass spectroscopy (ICP-MS). Additionally, for the first time, a heavy metal risk assessment strategy was used to evaluate the potential risks of traditional animal medicines by calculating estimated daily intake (EDI), target hazard quotient (THQ), and cancer risk (CR). To obtain a refined risk assessment, the frequency of exposure to traditional animal medicines was determined from questionnaire data, and the safe factor for TCM was applied. Based on the standard levels for leech, it was found that earthworm, hive, scorpion, and leech accumulated high levels of heavy metals. The combined THQ (cTHQ) values indicated that ingestion of most traditional animal medicines would not pose a risk to the health of either male or female human beings. However, it was indicated that attention should be paid to the potential risk associated with cicada slough, earthworm, scorpion, turtle shells, and hive. Among heavy metals, As and Hg contributed to a major extent to the risk to human health. The CR assessment for Pb and As indicated that, with the exception of earthworm, the cancer risk was less than the acceptable lifetime risk for both males and females. Owing to the higher body weight, both THQ and CR were generally lower for males than for females.


Assuntos
Arsênio/análise , Monitoramento Ambiental/métodos , Materia Medica/química , Medicina Tradicional Chinesa , Metais Pesados/análise , Animais , Feminino , Humanos , Masculino , Materia Medica/normas , Medicina Tradicional Chinesa/normas , Medição de Risco
17.
J Cell Mol Med ; 22(11): 5231-5243, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30589500

RESUMO

Hepatocellular carcinoma (HCC) is a highly prevalent cancer worldwide and it is necessary to discover and develop novel preventive strategies and therapeutic approaches for HCC. Herein, we report that EphrinB2 expression is correlated with liver cancer progression. Moreover, by using phosphorylated proteomics array, we reveal a pro-apoptosis protein whose phosphorylation and activation levels are up-regulated upon EphrinB2 knockdown. These results suggest that EphrinB2 may act as an anti-apoptotic protein in liver cancer cells. We also explored the therapeutic potential of HMQ-T-B10 (B10), which was designed and synthesized in our laboratory, for HCC and its underlying mechanisms in vitro and in vivo. Our data demonstrate that B10 could bind EphrinB2 and show inhibitory activity on human liver cancer cells. Moreover, induction of human liver cancer cell apoptosis by B10 could be augmented upon EphrinB2 knockdown. B10 inhibited HCC cell growth and induced HCC cell apoptosis by repressing the EphrinB2 and VEGFR2 signalling pathway. Growth of xenograft tumours derived from Hep3B in nude mice was also significantly inhibited by B10. Collectively, these findings highlight the potential molecular mechanisms of B10 and its potential as an effective antitumour agent for HCC.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Efrina-B2/genética , Neoplasias Hepáticas/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Am J Physiol Renal Physiol ; 315(2): F254-F262, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29384417

RESUMO

Salvianolic acid A (Sal A) has been shown to prevent and treat ischemic cardiovascular, as well as cerebral vascular diseases. However, little is known about Sal A in renal ischemia/reperfusion (I/R) injury. In this study, a renal I/R injury model in rats and a hypoxia/reoxygenation (H/R) model to damage proximal renal tubular cells (HK-2) were used to assess whether Sal A halts the development and progression of renal I/R injury. As compared with vehicle treatment, Sal A significantly attenuated kidney injury after renal I/R injury, accompanied by decreases in plasma creatinine, blood urea nitrogen levels, the number of apoptosis-positive tubular cells, and kidney oxidative stress. Sal A also activated phosphorylated protein kinase B (p-Akt) and phosphorylated-mammalian target of rapamycin (p-mTOR) compared with vehicle-treated I/R injury rats. In H/R-injured HK-2 cells, Sal A can reduce the levels of reactive oxygen species in a dose-related manner. Similar to the results from in vivo experiments, in vitro Sal A also increased the protein expression of phosphorylated-eukaryotic initiation factor 4E binding protein 1 (p-4EBP1) compared with vehicle. Furthermore, the cytoprotective activity of Sal A was inhibited by LY294002 and rapamycin. These findings indicate that Sal A can ameliorate renal I/R injury and promote tubular cell survival partly via the Akt/mTOR/4EBP1pathway. Sal A could be a candidate compound to prevent ischemic tissue damage.


Assuntos
Injúria Renal Aguda/prevenção & controle , Alcenos/farmacologia , Proteínas de Transporte/metabolismo , Rim/efeitos dos fármacos , Fosfoproteínas/metabolismo , Polifenóis/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fármacos Renais/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Injúria Renal Aguda/enzimologia , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Nitrogênio da Ureia Sanguínea , Linhagem Celular , Creatinina/sangue , Citoproteção , Modelos Animais de Doenças , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Rim/enzimologia , Rim/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Ratos Sprague-Dawley , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia
19.
Eur J Immunol ; 47(11): 1949-1958, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28688196

RESUMO

Mast cells are unique immune cells that function as sentinels in host defence reactions, including immediate hypersensitivity responses and allergic responses. The mast cell-specific receptor named MAS-related G protein-coupled receptor X2 (MRGPRX2) triggers mast-cell degranulation, a key process in anaphylactoid reactions. It is widely observed that antimicrobials can induce pseudo-allergic reactions (i.e. IgE-independent mechanism) with symptoms ranging from skin inflammation to life-threatening systemic anaphylaxis. However, their direct involvement and the mechanisms underlying anaphylactoid reactions caused by antimicrobials have not been demonstrated. Structurally different antimicrobials were screened by Ca2+ imaging using MRGPRX2 overexpressing HEK293 cells. MRGPRX2 related anaphylactoid reactions induced by these components were investigated by body temperature drop and mast cell degranulation assays. We showed that MRGPRX2 is involved in allergic-like reactions to three types of antimicrobials in a dose-dependent manner. However, mast cells lacking the receptor show reduced degranulation. Furthermore, mice without MAS-related G protein-coupled receptor B2 (the orthologous gene of MRGPRX2) exhibited reduced substance-induced inflammation. Interestingly, ß-lactam and antiviral nucleoside analogues did not induce anaphylactic reactions, which were also observed in vitro. These results should alarm many clinicians that such drugs might induce anaphylactoid reactions and provide guidance on safe dosage of these drugs.


Assuntos
Anafilaxia/induzido quimicamente , Anti-Infecciosos/toxicidade , Degranulação Celular/efeitos dos fármacos , Hipersensibilidade a Drogas/imunologia , Mastócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores de Neuropeptídeos/imunologia , Animais , Anti-Infecciosos/imunologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
20.
Electrophoresis ; 39(9-10): 1181-1189, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29359345

RESUMO

Traditional Chinese medicine (TCM) has been used for prevention and treatment of various diseases for many decades. TCM injection is a new dosage form, with incidence of anaphylactoid reactions increasing every year. In this study, the rat basophilic leukemia 2H3 (RBL-2H3) and laboratory of allergic disease 2 (LAD2) dual-mixed/CMC was established and was coupled with an HPLC-ESI-IT-TOF-MS system to identify the potential allergenic components in Haqing injection. Cinobufagin, piperine, osthole, praeruptorin A, and schizandrin A were screened from Haqing injection via this coupled system. Competitive binding assay showed piperine, praeruptorin A, and schizandrin A acting on MrgprX2 and cinobufagin and osthole act on the IgE receptor. The release of mediators of anaphylaxis results showed cinobufagin and osthole can cause anaphylactoid reactions by triggering the release of ß-hexosaminidase and histamine via IgE-R. Praeruptorin A and schizandrin A could promote the release of ß-hexosaminidase and histamine via MrgprX2 receptor. In summary, the dual-mixed/CMC model can significantly improve the efficiency of target component identification from a complex sample. When combined with competitive binding assay and validation of biological activities, this model enables accurate determination of the dual-target components, offering improved methods for quality control of TCM injections.


Assuntos
Alérgenos/análise , Medicamentos de Ervas Chinesas/análise , Leucemia/tratamento farmacológico , Espectrometria de Massas em Tandem/métodos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Hipersensibilidade , Medicina Tradicional Chinesa , Ratos
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