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Gliomas are the most common primary intracranial tumor worldwide. The maintenance of telomeres serves as an important biomarker of some subtypes of glioma. In order to investigate the biological role of RTEL1 in glioma. Relative telomere length (RTL) and RTEL1 mRNA was explored and regression analysis was performed to further examine the relationship of the RTL and the expression of RTEL1 with clinicopathological characteristics of glioma patients. We observed that high expression of RTEL1 is positively correlated with telomere length in glioma tissue, and serve as a poor prognostic factor in TERT wild-type patients. Further in vitro studies demonstrate that RTEL1 promoted proliferation, formation, migration and invasion ability of glioma cells. In addition, in vivo studies also revealed the oncogene role of RTEL1 in glioma. Further study using RNA sequence and phospho-specific antibody microarray assays identified JNK/ELK1 signaling was up-regulated by RTEL1 in glioma cells through ROS. In conclusion, our results suggested that RTEL1 promotes glioma tumorigenesis through JNK/ELK1 cascade and indicate that RTEL1 may be a prognostic biomarker in gliomas.
Assuntos
Neoplasias Encefálicas , Glioma , Humanos , Glioma/patologia , Neoplasias Encefálicas/genética , Transformação Celular Neoplásica/genética , Oncogenes , Biomarcadores , Proliferação de Células , Proteínas Elk-1 do Domínio ets/genética , DNA Helicases/genéticaRESUMO
Influenza A virus (IAV) H1N1 infection remains great challenge to public health and causes great burden over the world. Although there are anti-viral agents available, searching for effective agents to treat H1N1 infection is still in urgent because of the emergence of resistant strain. Protocatechuic acid (PCA) is a biological agent with multiple functions. In present study, we explored the effects of PCA on H1N1 infection. Mice infected with mouse adapted influenza strain A/Font Monmouth were administrated with PCA. The body weight change, mortality, lung index, viral titer, immune cell infiltration, and cytokine production in the lung were monitored. The activation of toll-like receptor 4 (TLR4) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway was investigated. PCA treatment prevented H1N1 infection-induced mice body weight loss and death. PCA reduced the lung index, viral titer, infiltration of immune cells, and cytokine level in the lung, as well as suppressed H1N1-induced TLR4/NF-κB activation. PCA protects mice against H1N1 infection and could be a potential therapeutic agent to treat influenza.
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Hidroxibenzoatos , Vírus da Influenza A Subtipo H1N1 , Infecções por Orthomyxoviridae , Animais , Modelos Animais de Doenças , Hidroxibenzoatos/farmacologia , Hidroxibenzoatos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/prevenção & controle , Ativação Transcricional/efeitos dos fármacosRESUMO
RATIONALE: A series of photodegradation impurities and a series of degradation impurities produced in autoclaving in xinfujunsu injection were discovered, and these unknown impurities were separated and characterized thoroughly using liquid chromatography tandem quadrupole time-of-flight mass spectrometry. METHODS: The column was a Platisil 5 µm ODS (4.6 × 250 mm, 5 µm). For the analysis of degradation impurities caused by light irradiation and autoclaving, the mobile phase was composed of 0.01 M ammonium formate aqueous solution and acetonitrile/isopropanol (90:10, V/V). Full scan LC-MS and LC-MS2 was carried out to obtain as much structural information as possible. The fragmentation behavior of actinomycin D, actinomycin S3 , and its impurities was studied and used to obtain information about the structures of these impurities. RESULTS: Based on MS2 spectral data and exact mass measurements, the chemical structures of two series of degradation impurities were characterized, among which five unknown impurities were photodegradation impurities and seven unknown impurities were degradation impurities produced in autoclaving of xinfujunsu injection. CONCLUSIONS: Based on characterization of impurities, this study also revealed the cause of impurity production and provided guidance for enterprises to improve the process and drug packaging material to reduce impurity content. Furthermore, this study also provided scientific basis for further improvement of official monographs in pharmacopoeias.
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Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/efeitos da radiação , Espectrometria de Massas em Tandem/métodos , Dactinomicina/análogos & derivados , Dactinomicina/química , Contaminação de Medicamentos , Medicamentos de Ervas Chinesas/isolamento & purificação , Temperatura Alta , Luz , FotóliseRESUMO
Treatments against influenza A viruses (IAV) have to be updated regularly due to antigenic drift and drug resistance. Poly (ADP-ribose) polymerases (PARPs) are considered effective therapeutic targets of acute lung inflammatory injury. This study aimed to explore the effects of PARP-1 inhibitor olaparib on IAV-induced lung injury and the underlying mechanisms. Male wild-type C57BL/6 mice were intranasally infected with IAV strain H1N1 to mimic pneumonia experimentally. Olaparib at different doses was intraperitoneally injected 2 days before and 5 consecutive days after virus stimulation. On day 6 post-infection, lung tissues as well as bronchoalveolar lavage fluid (BALF) were sampled for histological and biochemical analyses. Olaparib increased the survival rate of IAV mice dose-dependently. Olaparib remarkably reduced IAV mRNA expression, myeloperoxidase (MPO) level, and inflammatory cell infiltration in IAV lungs. Moreover, olaparib significantly reduced the level of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, and IL-4 and increased IL-10 in IAV lungs. Also, olaparib efficiently reduced IL-6, monocyte chemotactic protein (MCP)-1, granulocyte colony-stimulating factor (G-CSF), TNF-α, chemokine (C-X-C motif) ligand (CXCL)1, CXCL10, chemokine (C-C motif) ligand (CCL)3, and regulated on activation, normal T cell expressed and secreted (RANTES) release in IAV BALF. Olaparib decreased PARylated protein content and p65, IκBα phosphorylation in IAV lung tissues. This study successfully constructed the pneumonia murine model using IAV. Olaparib decreased IAV-induced mortality in mice, lung injury, and cytokine production possibly via modulation of PARP-1/NF-κB axis.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Pneumonia Viral/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Modelos Animais de Doenças , Humanos , Influenza Humana/virologia , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ftalazinas/administração & dosagem , Ftalazinas/uso terapêutico , Piperazinas/administração & dosagem , Piperazinas/uso terapêutico , Pneumonia Viral/virologia , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagemRESUMO
RATIONALE: Although the identification of degradation products of cefpirome sulfate has been reported, there has been no report concerning the impurities in bulk samples of this compound. To meet the requirements of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use, the structures of impurities whose content are over 0.1% need to be confirmed. Thus, characterization of the impurities in cefpirome sulfate bulk samples is critical for controlling the production of this drug. METHODS: The structures of cefpirome sulfate impurities were investigated using two-dimensional liquid chromatography (LC) coupled to electrospray ionization tandem mass spectrometry. In the first LC dimension, a Kromasil 100-5C18 column (4.6 mm × 250 mm, 5 µm) was used, and the mobile phases were 0.03 M ammonium dihydrogen phosphate solution and acetonitrile. In the second dimension, the column was a Shimadzu Shim-pack GISS C18 column (50 mm × 2.1 mm, 1.9 µm), and the mobile phases were 10 mM ammonium formate solution and methanol. An ion trap time-of-flight mass spectrometer operated in both positive and negative ion mode was employed in this study. RESULTS: Nine impurities and isomers in cefpirome sulfate, eight of which were previously unknown, were separated and characterized. Structures were proposed for the eight unknown compounds based on the MSn fragmentation data. The degradation behavior of cefpirome sulfate was also studied. CONCLUSIONS: Based on the characterization of impurities and isomers, this study could be used to improve the quality control of the cefpirome sulfate drug recommended in pharmacopoeias. The degradation behavior of cefpirome sulfate provides a basis for the selection of storage conditions.
Assuntos
Cefalosporinas/química , Oximas/química , Cromatografia Líquida de Alta Pressão/métodos , Contaminação de Medicamentos , Armazenamento de Medicamentos , Isomerismo , Espectrometria de Massas em Tandem , CefpiromaRESUMO
RATIONALE: The structural identification of impurities in cephalosporins has been reported. However, to the best of our knowledge, there was no report on the impurities of cefsulodin sodium, which is necessary for the quality control. Thus, the aim of this study was to separate and characterize the impurities in cefsulodin sodium raw material using liquid chromatography/tandem mass spectrometry (LC/MS/MS). METHODS: The analytes were separated on a Kromasil 100-5C18 column (4.6 mm × 250 mm, 5 µm) using a gradient elution with a mobile phase consisting of 1% ammonium sulphate aqueous solution and acetonitrile in the first dimension. The separation in the second dimension was carried on a Shimadzu Shim-pack GISS C18 column (50 mm × 2.1 mm, 1.9 µm) with a mobile phase consisting of 10 mM ammonium formate solution and methanol. RESULTS: The fragmentation behaviors of cefsulodin and its impurities were studied and the structures of the impurities were deduced based on the MSn data. The structures of ten unknown impurities were proposed based on the work carried out in this study. The degradation behaviors of cefsulodin sodium were also studied. This revealed that cefsulodin sodium should be stored in a dry, cool and dark closed container. CONCLUSIONS: Based on the characterization of impurities, this study not only revealed the mechanism by which impurities were produced, thus providing guidance to pharmaceutical companies for manufacturing process improvement and impurity control, but also provided a scientific basis for further improvement of official monographs in pharmacopoeias.
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RATIONALE: According to the requirements of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), the structures of impurities in pharmaceutical products present at over 0.1% need to be confirmed. Therefore, the aim of this study is to separate and identify the impurities in cephapirin sodium drug substances, so as to guide the industry to improve the production process and storage conditions and reduce the amount of impurities in the product. METHODS: In the first chromatography dimension, a Boston Green ODS (4.6 mm × 250 mm, 5 µm) column was used, with a mobile phase composed of 0.05 M sodium dihydrogen phosphate aqueous solution and acetonitrile. In the second dimension, the column was a Shimadzu Shim-pack GISS C18 (50 mm × 2.1 mm, 1.9 µm), using 10 mM ammonium formate solution and methanol as the mobile phase. RESULTS: The fragmentation behavior of cephapirin and its impurities and isomers was studied and the structures of impurities were deduced based on the MSn data. For six unknown impurities tentative structures were proposed. The degradation behavior of cephapirin sodium was also studied. Impurities 1 to 11 were found in commercial cephapirin sodium samples, indicating that cephapirin sodium should be stored in closed containers. CONCLUSIONS: The contradiction between the non-volatile mobile phase and mass spectrometry was solved by means of multiple heart-cutting approaches and an on-line desalting technique. Twelve impurities and isomers were separated and characterized. These results could be used to improve the methods described in pharmacopoeias for the quality control of cephapirin sodium.
RESUMO
RATIONALE: A new high-performance liquid chromatography method was developed for the determination of impurities in rutin tablets to improve on the method of the official monograph in national drug standards. Five impurities in rutin tablets were characterized using trap-free two-dimensional liquid chromatography coupled with ion trap/time-of-flight mass spectrometry (2D-LC/IT-TOFMS) in both positive and negative ion modes of electrospray ionization. METHODS: In the first dimension, the LC column was a Thermo Acclaim 120™ C18 (4.6 mm × 250 mm, 5 µm), and the mobile phase was composed of 0.1 M sodium dihydrogen phosphate aqueous solution (pH adjusted to 4.4 with phosphoric acid) and acetonitrile (80:20, v/v). In the second dimension, the column was a Shimadzu Shim-pack GISS C18 (50 mm × 2.1 mm, 1.9 µm), and the mobile phase was composed of 10 mM ammonium formate solution and methanol. RESULTS: The structures of five impurities in rutin tablets were deduced based on the MS n data in both positive and negative ion modes, in which two impurities were unknown. Impurity 1, impurity 2 and impurity 3 were proposed as flavonol 3,7-di-O-glycoside, flavonol mono-O-triglycoside and quercetin 3-O-glycoside, respectively, and impurity 4 and impurity 5 were proposed as kaempferol 3-O-rhamnosylglucoside and isorhamnetin 3-O-rhamnosylglucoside, respectively. CONCLUSIONS: The method established in this study was simple and reliable for the routine quality control of rutin tablets. The contradiction between non-volatile salt mobile phase and mass spectrometry was solved by means of a multiple heart-cutting 2D-LC approach and on-line desalination technology. Five impurities were separated and characterized. These results provide a scientific basis for further improving the national drug standard of rutin tablets.
Assuntos
Anti-Inflamatórios/química , Cromatografia Líquida de Alta Pressão/métodos , Contaminação de Medicamentos , Espectrometria de Massas/métodos , Rutina/química , ComprimidosRESUMO
BACKGROUND Insomnia seriously affects people's health and quality of life. Short-term use of Western drugs may also be harmful. Traditional Chinese medicine has been widely used to treat diseases in world. Therefore, this paper aims to study the therapeutic effect of berberine based on the insomnious rat model. MATERIAL AND METHODS The insomnia rat model was established by intragastric administration of caffeine and parachlorophenylalanine (PCPA). Berberine and diazepam were used to treat the established insomnia rats. Then, the pathological changes of insomnia rats were detected. In addition, transcriptome sequencing and data analysis were carried out using rat hippocampus. The expression of key genes was verified by quantitative polymerase chain reaction and western blot. RESULTS After 7 days of intragastric administration of berberine, the body weight, memory, and sleep quality of insomnia rats were significantly improved. The key roles of Erbb4, Erbb2, Ar, and Grin2a in berberine treatment were identified. Through the analysis of biological functions and signaling pathways, berberine was shown to play a salutary role through nervous system development and ErbB signaling pathway. Gene-set enrichment analysis (GSEA) results showed that berberine treatment affected more metabolic pathways. Compared with diazepam, berberine can play a faster role, and also improve the overall health level of insomnia rats. CONCLUSIONS These results suggest that berberine can alleviate insomnia in rats through a neuroprotective effect and improved metabolic level. Berberine has great potential in treatment of insomnia and might have better clinical significance.
Assuntos
Berberina/farmacologia , Distúrbios do Início e da Manutenção do Sono/tratamento farmacológico , Animais , Berberina/metabolismo , Modelos Animais de Doenças , Masculino , Medicina Tradicional Chinesa , Memória , Redes e Vias Metabólicas , Qualidade de Vida , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/metabolismo , Receptor ErbB-4/metabolismo , Receptores Androgênicos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
With overuse of the broad-spectrum antibiotics, the pulmonary fungal infection increasingly becomes the most common complication associated with senile pulmonary tuberculosis (TB) and attracts intensive attentions from clinicians. Here we presented the retrospective analysis of impact of fluconazole treatment on the clinical outcome and immune response in fungal co-infected tuberculosis patients. A randomized, double-blind, placebo-controlled trial of fluconazole (100â¯mg per day for consecutive weeks) in fungal-positive senile tuberculosis patients was conducted in our hospital. Peripheral eosinophil counts were computed by the automatic hematology analyzer. The secretory inflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α and chemokines chemokine C-X-C motif ligand (CXCL)9, CXCL10, CXCL11 were determined with enzyme-linked immunosorbent assay kits. The peripheral T helper 1â¯cells (Th1) and regulatory T cells (Treg) population were analyzed by flow cytometry. None of significant difference in respect to baseline TB score was observed between placebo and fluconazole groups. Administration of fluconazole significantly stimulated eosinophils population and secretion of inflammatory cytokines IFN-γ and TNF-α. Simultaneously, the peripheral Th1% and chemokines including CXCL9, CSCL10, CXCL11 were markedly induced in response to fluconazole treatment. Fungal infection significantly affected host immunity during tuberculosis which was effectively reversed by fluconazole treatment.
Assuntos
Coinfecção , Fluconazol/imunologia , Fluconazol/uso terapêutico , Micoses/tratamento farmacológico , Micoses/imunologia , Tuberculose/tratamento farmacológico , Tuberculose/imunologia , Idoso , Idoso de 80 Anos ou mais , Índice de Massa Corporal , Quimiocina CXCL10/sangue , Quimiocina CXCL11/sangue , Quimiocina CXCL9/sangue , Quimiocinas/sangue , China , Citocinas/sangue , Método Duplo-Cego , Eosinófilos , Fluconazol/administração & dosagem , Fluconazol/farmacologia , Humanos , Interferon gama/sangue , Masculino , Pessoa de Meia-Idade , Micoses/complicações , Estudos Retrospectivos , Células Th1 , Resultado do Tratamento , Tuberculose/complicações , Fator de Necrose Tumoral alfa/sangueRESUMO
It is well known that stressful situation is one of the important factors causing insomnia, however, the underlying mechanism is still elusive. Therefore, the establishment of a suitable animal model of stress insomnia will be of great help to solve this problem. In this study, by combining with chronic unpredictable stress (multitude of stressors) and sleep deprivation, we attempted to establish a rat model of stress insomnia. It was observed that rats with stress insomnia showed significant weight loss, and less sleep quality in pentobarbital sodium induced sleep test and electroencephalogram detection. Moreover, rats with stress insomnia showed greater depression and anxiety detected by forced swimming, sucrose preference test and open field. Since oxidative stress has been reported to be involved in insomnia, we further evaluated the production of oxidative stress and found that the levels of lipid peroxidation product malondialdehyde (MDA) in liver, serum total bilirubin and urine biopyrrin were all significantly increased in rats with stress insomnia. In addition, we also found that the memory of these rats with stress insomnia was also obviously reduced in water maze. Taken together, these results demonstrate that the emotional behaviors, memory, oxidative and metabolism of the rats were all significantly changed after modeling, indicating a rat model of stress insomnia was successful establishment, and this animal model will provide basis to further explore the underlying mechanism of chronic stress in insomnia.
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Solute carrier family 39 member 10 (SLC39A10) belongs to a subfamily of zinc transporters and plays a key role in B-cell development. Previous studies have reported that its upregulation promotes breast cancer metastasis by enhancing the influx of zinc ions (Zn2+); however, its role in gastric cancer remains totally unclear. Here, we found that SLC39A10 expression was frequently increased in gastric adenocarcinomas and that SLC39A10 upregulation was strongly associated with poor patient outcomes; in addition, we identified SLC39A10 as a direct target of c-Myc. Functional studies showed that ectopic expression of SLC39A10 in gastric cancer cells dramatically enhanced the proliferation, colony formation, invasiveness abilities of these gastric cancer cells and tumorigenic potential in nude mice. Conversely, SLC39A10 knockdown inhibited gastric cancer cell proliferation and colony formation. Mechanistically, SLC39A10 exerted its carcinogenic effects by increasing Zn2+ availability and subsequently enhancing the enzyme activity of CK2 (casein kinase 2). As a result, the MAPK/ERK and PI3K/AKT pathways, two major downstream effectors of CK2, were activated, while c-Myc, a downstream target of these two pathways, formed a vicious feedback loop with SLC39A10 to drive the malignant progression of gastric cancer. Taken together, our data demonstrate that SLC39A10 is a functional oncogene in gastric cancer and suggest that targeting CK2 is an alternative therapeutic strategy for gastric cancer patients with high SLC39A10 expression.
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Neoplasias Gástricas , Animais , Camundongos , Caseína Quinase II/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sistema de Sinalização das MAP Quinases , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Zinco/metabolismo , HumanosRESUMO
To effectively control the polymerized impurities in mezlocillin sodium and sulbenicillin sodium, a HPSEC method with TSK-gel G2000SWxl column and a RP-HPLC method with C18 analytical column were established to replace the classical gel filtration chromatography with Sephadex G-10 gel as stationary phase. By studying the chromatographic behavior of polymerized impurities in both methods with different chromatographic separation mechanisms, the polymerized impurities in mezlocillin sodium and sulbenicillin sodium were separated and detected effectively. The column switching two-dimension liquid chromatography ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities eluted from the HPSEC method and RP-HPLC method for both drugs. The structures of the polymerized impurities in mezlocillin sodium and sulbenicillin sodium were deduced based on the MSn data. The results showed that the polymerized impurities detected by HPSEC method and RP-HPLC method were completely different. Therefore, two methods should be used meanwhile to control the polymerized impurities in mezlocillin sodium and sulbenicillin sodium.
Assuntos
Preparações Farmacêuticas , Sulbenicilina , Cromatografia Líquida de Alta Pressão , Contaminação de Medicamentos , Mezlocilina , Sódio , Espectrometria de Massas em TandemRESUMO
Polymerized impurities in ß-lactam antibiotics can induce allergic reaction, which seriously threaten the health of patients. In order to study the polymerized impurities in cefotaxime sodium and cefepime, a HPSEC method with TSK-gel G2000SWxl column and a RP-HPLC method with C18 column were established to replace the classical gel filtration chromatography with Sephadex G-10 gel as stationary phase. Two-dimensional (2D) liquid chromatography was employed to further investigate the HPSEC and RP-HPLC method and ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities eluted from the two methods. The structures of the polymerized impurities in cefotaxime sodium and cefepime were deduced based on the MSn data, six of which were previously unknown. The corresponding relationship of impurities between the two methods was established, and the specificity of the two methods was compared. The results showed that the polymerized impurities in cefotaxime sodium and cefepime were co-eluted with other small molecular weight impurities with high polarity in HPSEC, leading to a poor specificity. The newly established RP-HPLC methods could effectively separate and detect polymerized impurities and were suitable for the quality control in daily analysis.
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Cefotaxima , Contaminação de Medicamentos , Cefepima , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Cromatografia Gasosa-Espectrometria de Massas , HumanosRESUMO
Background: Insomnia is a clinical problem of significant public health importance; however, the underlying pathogenesis of this disorder is not comprehensively understood. Methods: To identify potential treatment targets and unfold one of the gaps that were involved in insomnia pathological mechanisms, we employed a tandem mass tag-based (TMT) quantitative proteomics technology to detect differentially expressed proteins (DEPs) in serum from patients with insomnia and controls. DEPs were further analyzed by bioinformatics platforms. In addition, parallel reaction monitoring (PRM) was used to verify the TMT results. Results: Patients with insomnia had poorer sleep quality compared with healthy controls. A total of 106 DEPs were identified among patients with insomnia and controls. They were mainly enriched in immune and inflammation-related biological functions and signaling pathways. Using the protein-protein interaction network, we screened the 10 most connected proteins as key DEPs. We predicted that four key DEPs were subject to targeted regulation by natural compounds of herbs. Eight key DEPs were validated using PRM in an additional 15 patients with insomnia and 15 controls, and the results also supported the experimental findings. Conclusion: We identified aberrantly expressed proteins in insomnia that may be involved in the immune-inflammatory response. The 10 key DEPs screened may be potential targets for insomnia, especially FN1, EGF, HP, and IGF1. The results of this study will broaden our understanding of the pathological mechanisms of insomnia and provide more possibilities for pharmacotherapy.
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To effectively control the polymerized impurities in cefmetazole sodium, novel high performance gel filtration chromatography (HPSEC) with TSK-gel G2000SWxl column and RP-HPLC method with C18 column were used in replace of classical gel filtration chromatography with Sephadex G-10 gel. By studying the chromatographic behavior of polymerized impurities in both chromatographic systems with different chromatographic separation principles, the polymerized impurities in cefmetazole sodium were separated and detected effectively. The two-dimensional liquid chromatography tandem ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) was applied to characterize the structures of polymerized impurities eluted from HPSEC method, and liquid chromatography tandem ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities and other unknown impurities eluted from RP-HPLC method. The structures of fourteen unknown impurities in cefmetazole sodium were deduced based on the MS n data, nine of which were polymerized impurities. The corresponding relationship between impurities in the HPSEC method and RP-HPLC method was established, and the specificity of the two methods was evaluated. The RP-HPLC method for analysis of the polymerized impurities has higher column efficiency and specificity than the HPSEC method. The RP-HPLC method is suitable for quality control of the polymerized impurities in cefmetazole sodium. The forming mechanisms of degradation impurities in cefmetazole sodium were also studied.
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Cefmetazol , Contaminação de Medicamentos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Espectrometria de MassasRESUMO
Background: Insomnia is a sleep disorder and the cause of many healthy problems. However, there are few studies on patients with insomnia and dreaminess at present. Therefore, this study is aimed at exploring the pathological molecular mechanisms and potential diagnostic and therapeutic targets related to insomnia patients with more dreams. Methods: Sleep characteristics of 36 primary insomnia patients with more dreams and 36 well sleeping participants were assessed using polysomnography (PSG) and Pittsburgh Sleep Quality Index (PSQI). Serum samples from 9 insomnia patients and 9 controls were randomly selected for proteomic detection. Differentially expressed proteins (DEPs) between the two groups were identified; enrichment analysis and PPI network were performed. The top 10 most connected proteins in the PPI network were subjected to targeted drug prediction and screened key proteins. Proteins with targeted drugs were recognized as key proteins and subjected to ELISA detection. Results: Insomnia patients had a distinct REM behavior disorder signature compared with controls. Proteomic sequencing identified 76 DEPs. Enrichment analysis found that DEPs were significantly enriched in the complement and coagulation cascades. Metabolic responses were also activated in insomnia patients. Among the hub proteins screened in the PPI network, APOA1, APOB, F2, and SPARC may be targeted by many herbal medicines and considered as key proteins. ELISA assays validated their differential expression between insomnia and controls. Conclusion: In this study, we identified the potential key proteins of insomnia patients with more dreams. The pathological process may associate with inflammation and metabolic response. These results provide molecular targets for diagnostic and therapeutic targets. The results of our analysis suggest that the expression changes of key proteins have a good predictive diagnostic role for the occurrence of insomnia with more dreams in patients.
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Distúrbios do Início e da Manutenção do Sono , Humanos , Polissonografia , Proteômica , SonoRESUMO
Josamycin and midecamycin are consisted of three groups of components with different ultraviolet maximum absorption wavelengths (λmax), which are 231 nm, 280 nm and 205 nm. The quantitative analysis of all these components is challengeable due to the absence of the respective reference substances. To address this problem, universal and reliable methods were developed using high performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) for the quantitative analysis of components in josamycin and midecamycin. The chromatographic conditions and CAD parameters setting were optimized. Subsequently, the components were identified using HPLC coupled with ion trap/time-of-flight mass spectrometry (IT/TOF MS). The developed methods were validated by assessing linearity, limit of quantitation (LOQ), accuracy, precision and robustness. Good separations were achieved for all components and the adjustment of the filter valve and power function value efficiently improved sensitivity. The developed methods were more comprehensive than current HPLC-UV method. The experimental results demonstrated good linearity with coefficients of determination (R2) greater than 0.999 in the range of 0.002-0.30 mg mL-1. The limits of detection (LOD) were ranging from 1.8 to 2.0 µg·mL-1. The intra-day and inter-day RSD values were less than 2.0 % (n = 6) and 5.6 % (n = 9) respectively. The recoveries were 95.0 %-124.0 % at the spiked concentration levels of 0.05 %, 0.50 %, 0.10 % and 2.5 % with relative standard deviations (RSDs, n = 3) lower than 2.0 %. Finally, the developed methods were successfully applied to the quantitative analysis of minor components and used main components (leucomycin A3 and midecamycin A1) as alternative reference substance of minor components. The overall results demonstrated that the HPLC-CAD was a good alternative for the quantitative analysis of multi-components in 16-membered macrolides.
Assuntos
Josamicina , Leucomicinas , Aerossóis , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Reprodutibilidade dos TestesRESUMO
Toxic impurities were found in leucomycin and its preparation, however the content determination of impurities was challengeable due to the lacking of their reference standards. In this study, we developed high-performance liquid chromatography method coupled with charged aerosol detection (CAD) for the quantification of related substance of leucomycin (kitasamycin) bulk drugs and tablets, however, the CAD was not yet popular. In order to carry out quantitation work conveniently in the laboratory without CAD instruments, a high-performance liquid chromatography method coupled with ultraviolet (UV) detection was developed with the assistant of the HPLC-CAD results. The relative response of impurities on CAD chromatogram was used for guiding the establishment of HPLC-UV method, which could achieve the quantitation task in the absence of impurity reference standards. The developed HPLC-UV method was validated according to the ICH guideline and showed good precision, reproducibility and linearity with determination coefficient higher than 0.9999. The limit of detection and quantitation were 0.3 and 0.5 µg mL-1, respectively. The recoveries were 92.9 %-101.5 % at the spiked concentration levels of 0.1 %, 0.8 %, 1.0 and 1.2 % with relative standard deviations (RSDs, n = 3) lower than 2.0 %. Finally, the developed HPLC-CAD and -UV methods were compared by the determination of impurities in several batches of leucomycin bulk drugs and tablets. The results demonstrated that the developed HPLC-UV method was simple and reliable. This study developed methods to quantify the related substance in leucomycin and tablets, and discussed a strategy of the conversion of HPLC-CAD method to HPLC-UV method. The developed methods could be considered for implementation into pharmacopeial monographs in the future.
Assuntos
Kitasamicina , Aerossóis , Cromatografia Líquida de Alta Pressão , Reprodutibilidade dos Testes , Espectrofotometria Ultravioleta , ComprimidosRESUMO
BACKGROUND: Insomnia is an economic burden and public health problem. This study is aimed at exploring potential biological pathways and protein networks for insomnia characterized by wakefulness after sleep. METHOD: Proteomics analysis was performed in the insomnia group with wakefulness and the control group. The differentially expressed proteins (DEPs) were enriched; then, hub proteins were identified by protein-protein interaction (PPI) network and verified by parallel reaction monitoring (PRM). RESULTS: Compared with the control group, the sleep time and efficiency of insomnia patients were decreased, and awakening time and numbers after sleep onset were significantly increased (P < 0.001). The results of proteomic sequencing found 68 DEPs in serum under 1.2-fold changed standard. These DEPs were significantly enriched in humoral immune response, complement and coagulation cascades, and cholesterol metabolism. Through the PPI network, we identified 10 proteins with the highest connectivity as hub proteins. Among them, the differential expression of 9 proteins was verified by PRM. CONCLUSION: We identified the hub proteins and molecular mechanisms of insomnia patients characterized by wakefulness after sleep. It provided potential molecular targets for the clinical diagnosis and treatment of these patients and indicated that the immune and metabolic systems may be closely related to insomnia characterized by wakefulness after sleep.