Frontiers in Chemistry
·
2020
·
IF 3.693
Wang Yao,Li Zhe,Yu Hanyang
Selective protein recognition is critical in molecular biology techniques such as Western blotting and ELISA. Successful detection of the target proteins in these methods relies on the specific interaction of the antibodies, which often bring a high production cost and require a long incubation time. Aptamers represent an alternative class of simple and affordable affinity reagents for protein recognition, and replacing antibodies with aptamers in Western blotting would potentially be more time- and cost-effective. In this work, multiple fluorescent DNA aptamers were isolated by in vitro selection to selectively label commonly used tag proteins including GST, MBP, and His-tag. The generated aptamers G1, M1, and H1 specifically bound to their cognate target proteins with nanomolar affinities, respectively. Compared with conventional antibody-based immunoblotting, such aptamer-based procedure gave a cleaner background and was able to selectively label target protein in a complex mixture. Lastly, the identified aptamers were also effective in recognition of different fusion proteins with the same tag, thus greatly expanding the scope of the potential applications of these aptamers. This work provided aptamers as useful molecular tools for selective protein recognition in Western blotting analysis.
研究领域:
分子生物学
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生物化学
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DOI
PubMed
高影响因子
Biotechnology Journal
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2023
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IF 4.7
Yan-Fei Shen,Ming-Jie Liu,Zhu Long,Xiaobang Shi,Meng-Lu Liu
High-throughput western blot (WB) analysis can be used to obtain more consistent, comparable, and informative data from precious samples and materials with extremely limited availability, such as various age-related, subtype-specific human induced neurons (hiNs). In this study, p-toluenesulfonic acid (PTSA), an odorless tissue fixative, was used to inactivate horseradish peroxidase (HRP) and develop a high-throughput WB method. PTSA-treated blots demonstrated rapid and efficient HRP inactivation without detectable protein loss or epitope damage. With a brief PTSA treatment (1 min at room temperature [RT]) before every subsequent probing, 10 dopaminergic hiN proteins could be sequentially, sensitively, and specifically detected in the blot. The resulting WB data confirmed the age-associated and neuron-specific features of hiNs and revealed a significant reduction in two Parkinson's disease-associated proteins, UCHL1 and GAP43, in normal aging dopaminergic neurons. Overall, this study developed a unique and high-efficiency WB analysis method for capturing robust and useful data from limited, precious samples. Graphical and Lay Summary Inactivation of horseradish peroxidase (HRP) facilitates multiple reprobing of a single blot to sequentially detect different proteins instead of rerunning several gels and blots. In this study, the authors demonstrate that p-toluenesulfonic acid (PTSA) inactivates HRP rapidly and efficiently, allowing simple, flexible, and high-efficiency western blot (WB) analysis of diverse proteins on a single blot for age-related, subtype-specific human induced neurons. The results imply the promising potential of PTSA as an excellent and user-friendly HRP-inactivating reagent for novel high-throughput WB analysis, with considerable importance for research on limited, precious samples.
研究领域:
神经科学
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分子生物学
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蛋白质组学
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DOI
PubMed
高影响因子
Scientific Reports
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2024
·Vol. 14
·期 1
·1-10
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IF 3.8
Sun Huiwen,Wei Moris,Guo Amber,Zhang Ci,Wang Yuefeng,Huang Renhui,Li Xiaoxiao,Zhan Jeffrey,Wu Jonny,Jiang Bruce
Conjugation to a carrier protein is essential to give rise to the antigenicity of hapten. Three carrier proteins e.g. KLH (Keyhole Limpet hemocyanin), BSA (bovine serum albumin), and OVA (Ovalbumin) were used mostly. KLH is advantageous to the others, majorly owing to its strong immunogenicity and limited usage in other biological assays. However, the cost of obtaining Keyhole Limpet is high and the solubility of KLH is not as well as the other carriers, especially after hapten conjugation. Here, we extracted the shrimp hemocyanin (SHC) from Litopenaeus vannamei (L. vannamei), which is a commonly sea product worldwide. The high pure SHC could be acquired by two-step purification, with a production yield of > 1 g proteins (98% pure) per 1 kg shrimp. Compared to KLH, the peptide-SHC conjugates exhibit higher solubility after hapten conjugation. Meanwhile, compared with KLH, SHC induces comparable antibody production efficiency in mammals, with or without conjugation. Furthermore, rabbit polyclonal antibodies or mouse monoclonal antibodies were generated by immunizing SHC-peptide conjugates, and the subsequent antibodies were confirmed to be used in western blot, immunofluorescence and immunohistochemistry. Therefore, we demonstrated that SHC may be used as a substitute for KLH in future antibody and vaccine development.
研究领域:
免疫学
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蛋白质化学
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生物技术
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DOI
PubMed
高影响因子
Chinese Journal of Integrative Medicine
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2022
·Vol. 29
·期 3
·244-252
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IF 2.626
Wang Yuan-chun,Hui Jian-rong,Xiao Gang,Ma Qiao-lin
Objective To investigate the mechanism of the effect of Astragalus membranaceus ( A. membranaceus ) on lung adenocarcinoma at the molecular level to elucidate the specific targets according to the network pharmacology approach. Methods The active components of A. membranaceus and their potential targets were collected from the Traditional Chinese Medicine Systems Pharmacology Database. Lung adenocarcinoma-associated genes were acquired based on GeneCards, Online Mendelian Inheritance in Man (OMIM), PharmGKB, and Therapeutic Targets databases. The PI3K/AKT signaling pathway-related genes were obtained using Reactome portal. Networks of “ingredient-target” and “ingredient-target-pathway-disease” were constructed using the Cytoscape3.6.0 software. The relationships among targets were analyzed according protein-protein interaction (PPI) network. Finally, molecular docking was applied to construct the binding conformation between active ingredients and core targets. Cell counting kit 8 (CCK8) and Western blot assays were performed to determine the mechanism of the key ingredient of A. membranaceus . Results A total of 20 active components and their 329 targets, and 7,501 lung adenocarcinoma-related genes and 130 PI3K/AKT signaling pathway-related genes were obtained. According to Venn diagram and PPI network analysis, 2 mainly active ingredients, including kaempferol and quercetin, and 6 core targets, including TP53, MAPK1, EGF, AKT1, ERBB2, and EGFR, were identified. The two important active ingredients of A. membranaceus , kaempferol and quercetin, exert the therapeutic effect in lung adenocarcinoma partly by acting on the 6 core targets (TP53, MAPK1, EGF, AKT1, ERBB2, and EGFR) of PI3K/AKT signaling pathway. Expressions of potential targets in lung adenocarcinoma and normal samples were analyzed by using UALCAN portal and found that ERBB2 was overexpressed in lung adenocarcinoma tissues and upregulation of it correlated with clinicopathological characteristics. Finally, quercetin repressed viabilities of lung adenocarcinoma cells by targeting ERBB2 on PI3K/AKT signaling confirmed by CCK8 and Western blot. Conclusion Our finding unraveled that an active ingredient of A. membranaceus , quercetin, significantly inhibited the lung adenocarcinoma cells proliferation by repressing ERBB2 level and inactivating the PI3K/AKT signaling pathway.
研究领域:
药理学
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肿瘤学
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分子生物学
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生物信息学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF ETHNOPHARMACOLOGY
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2024
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IF 4.8
Li-Ying Han,Hao Yu,Shuai Wang,Yong-Rui Bao,Tian-Jiao Li,Ying Zheng,Xi Luo,Meng-Nan Jia,Qiang Zhang,Xian-Sheng Meng
Ethnopharmacological relevance Colorectal cancer (CRC) belongs to the category of intestinal wind, anal ulcer, abdominal mass and other diseases in traditional Chinese medicine (TCM). Floris Sophorae Powder (F.S), is a classical prescription is recorded in Puji Benshi Fang for the treatment of intestinal carbuncle. It has been incorporated into the prescriptions for the treatment of intestinal diseases and achieved remarkable results in modern medicine. However, the mechanism of F.S in the treatment of colorectal cancer remains unclear and requires further study. Aim of the study To investigate F.S in treating CRC and clarify the underlying mechanism. Materials and methods This study was based on Dextran Sulfate Sodium Salt (DSS) combined with Azoxymethane (AOM) induced CRC mouse model to clarify the pharmacological effects of F.S. The serum metabolomics was used to study the mechanism of action, and the chemical composition of F.S was found by UPLC-Q-TOF-MS. The rationality of serm metabolomics results was verified through the clinical target database of network pharmacology, and the upstream and downstream targets of related pathways were found. The mechanism pathway was verified by Western blot to clarify its mechanism of action. Results In vivo pharmacological experiments showed that F.S inhibited tumor growth and improved hematochezia. The vital signs of mice in the high-dose F.S group approached to those in the control group. A total of 43 differential metabolites were found to be significantly changed by serum metabolomics. F.S could modulate and recover most of the differential metabolites, which proved to be closely related to the KRAS/MEK-ERK signaling pathway. A total of 46 compounds in F.S were identified, and the rationality of serm metabolic pathway was verified by network pharmacology. Western blot results also verified that the expression of KRAS, E2F1, p-MEK and p-ERK were significantly decreased after F.S treatment. Conclusion Classical prescription Floris Sophorae Powder treat colorectal cancer by regulating KRAS/MEK-ERK signaling pathway.
研究领域:
药理学
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肿瘤学
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中医
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代谢组学
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信号转导
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DOI
PubMed
高影响因子
BIOMEDICINE & PHARMACOTHERAPY
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2018
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IF 3.457
Li Zhang,Hui-Xia Li,Wu-Si Pan,Farhan Ullah Khan,Cheng Qian,Feng-Rong Qi-Li,Xiaojun Xu
Background and aims Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome and is characterized by steatosis, inflammation, and fibrosis. We aim to characterize the hepatoprotective effects of Leonurine hydrochloride (LH) and the possible pathway in a cell and rodent model of diet-induced steatohepatitis (NASH). Methods For in vitro studies, Palmitic acid (PA) and free fatty acid (FFA) induced HepG2 and HL7702 steatosis cell models were used. For in vivo studies, NASH was induced by feeding mice MCD diet. These mice received either placebo or LH at three different doses (50、100、200 mg/kg/day) for 6 weeks. Histological staining’s, and commercially available kits for ALT and AST and hepatic contents of TG, TC, MDA, SOD, and GSH were used to assess NASH. Furthermore, relative liver protein and gene expression levels were determined by Western Blot and qPCR, respectively. Results After establishing NASH models, LH treatment improved lipid accumulation, hepatic contents of TG, TC, and expression levels of ALT and AST in dose-dependent manner. Also, LH improved MDA, SOD, and GSH expression levels. The results of RT-PCR and Western blotting showed that LH upregulated the expression of AMPK phosphorylation and downregulated SREBP-1c and its target genes expression level. Conclusions Our data reveal the promising role of Leonurine hydrochloride in the prevention and treatment of NASH, in vitro and in vivo . This effect may be partially mediated by the AMPK/SREBP1 pathway. These findings provide a novel therapeutic target for the clinical treatment of NASH.
研究领域:
药理学
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肝病学
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代谢性疾病
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产品关联
DOI
PubMed
高影响因子
PHYTOMEDICINE
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2021
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IF 5.34
Shusheng Fan,Xiaoyun Liu,Yu Wang,Xueyang Ren,Yue Liu,Ying Dong,Qiqi Fan,Jing Wei,Jiamu Ma,Axiang Yu,Ruolan Song,Hong Sui,Meng Shen,Fang Fang,Qing Xia,Gaimei She
Background Thymus quinquecostatus Celak. has been widely used as a spice and a folk medicine for relieving exterior syndrome and alleviating pain in China. Purpose To explore the protective effects and the underlying mechanism against cerebral ischemia-reperfusion injury (CIRI) of the T. quinquecostatus combining with its chemical composition. Study design and methods High-polar extract (HPE) was extracted from T. quinquecostatus and polyphenols in HPE were enriched to obtain polyphenol-rich fraction (PRF) using Macroporous resin. The free radicals and zebrafish embryos were used to compare the antioxidant activities of HPE and PRF in vitro and in vivo . Then, the transient middle cerebral artery occlusion (tMCAO) model was established in rats. Neurological deficit score, infarction rate, morphology and apoptosis of neurons were examined to investigate the protective effects of PRF on CIRI. The mRNA and protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) and the activities of downstream antioxidant enzymes in ischemia tissues were determined to clarify the underlying mechanisms. Also, reactive oxygen species (ROS) level in zebrafish embryos were detected after incubation with PRF for a short time (2 h) to investigate whether PRF could directly eliminate free radicals. Finally, chemical composition of PRF were analyzed to investigate the material basis for antioxidant activity and anti-CIRI effect. Results Compared with HPE, PRF showed stronger antioxidant activities. PRF exhibited obvious protective effects including ameliorating neurological deficit, lowering infarction rate, and improving the cellular morphology in hippocampus CA1 and cortex after tMCAO. TUNEL staining suggested PRF dose-dependently improved the apoptosis of the neurons in ischemic cortex. RT-qPCR and Western Blot results suggested that PRF regulated oxidative stress (OS) via activating the Keap1/Nrf2/HO-1 signaling pathway . Also, PRF could directly scavenge excessive ROS in zebrafish embryos after a short-time PRF incubation. The anti-CIRI effect might be primarily attributed to the abundant polyphenols in PRF, including flavonoids , polymethoxylated flavonoids, flavonoid glycosides , and phenolic acids. Conclusion T. quinquecostatus contains abundant polyphenols and exhibited a good protective effect against CIRI via dual antioxidant mechanisms, providing a reference for further research and application for this plant.
研究领域:
药理学
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神经科学
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分子生物学
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植物科学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF ETHNOPHARMACOLOGY
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2021
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IF 4.36
Yuling Tong,Ruilin Wang,Xia Liu,Miao Tian,Yanling Wang,Yanfei Cui,Wenjun Zou,Yanling Zhao
Ethnopharmacological relevance Zuojin Pill (ZJP) is a classic prescription composed of Coptis chinensis and Tetradium ruticarpum (A.Juss.) T.G.Hartley, which is often used in the treatment of digestive system diseases . Aim of this study The purpose of this study was to explore the therapeutic effect and potential mechanism of ZJP on chronic atrophic gastritis (CAG) induced by MNNG . Materials and methods The GES-1 and rat model of CAG was established by MNNG. Detection of cell viability, morphological changes and proliferation of GES-1 by CCK-8 and high content screening (HCS) assay. G-17, IL-8 and TNF-α in rat serum were detected by ELISA kit. The expression of related mRNA and protein on TGF-β1/PI3K/Akt signal axis were detected by RT-PCR and Western blot . Results The results showed that ZJP could significantly improve the GES-1 damage induced by MNNG and improve the gastric histomorphology of CAG rats. The intervention of ZJP could significantly reduce the content of G-17 and inflammatory factors IL-8, TNF- α, IL-6 and IL-1β, inhibit the expression of TGF-β1, PI3K and their downstream signals p-Akt, p-mTOR, P70S6K, and promote the expression level of PTEN, LC3-II and Beclin-1. Conclusion ZJP has a good therapeutic effect on CAG induced by MNNG, which may be closely related to the inhibition of TGF-β1/PI3K/Akt signal pathway.
研究领域:
药理学
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民族药理学
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胃肠病学
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分子生物学
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细胞生物学
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产品关联
DOI
PubMed
高影响因子
Biomed Research International
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2019
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IF 2.197
Dongyu Xie,Ping Zhou,Lin Liu,Wenjing Jiang,Haina Xie,Liang Zhang,Donghao Xie
Suitable content of iron is essential for human body, but iron overload is associated with many kinds of diseases including chronic liver damage. Recently, researchers find that iron overload promotes hepatocyte autophagy and apoptosis. However, the mechanism of iron overload in liver damage remains unclear. In this study, Lo2 cells were selected as the research object, iron dextran was a model drug, and astragaloside IV was a therapeutic drug to explore the role of iron overload. MTT assay and Annexin/PI double staining were used to measure cell viability and apoptosis. Ultrastructure was observed by transmission electron microscopy. The expression levels of apoptosis and autophagy-related proteins were determined by real-time PCR and Western Blot. The results showed that iron dextran could significantly inhibit Lo2 cell viability and increase the apoptosis rate, while astragaloside IV could reverse the inhibition of Lo2 cell viability and decrease the apoptosis rate. Transmission electron microscopy showed a significant increase in the number of autophagosomes after administration of iron dextran, and the application of astragaloside IV reduced the production of autophagosomes. LC3II/I was significantly upregulated in the model group but decreased in the astragaloside IV treatment group, and P62 showed the opposite trend. Iron dextran significantly upregulated the expression of Bax and downregulated Bcl2, while astragaloside IV reversed this trend. Finally, the inhibition of hepcidin caused by iron dextran was counteracted by astragaloside IV. In conclusion, the experimental results show that the iron overload model mainly induces excessive autophagy and apoptosis of hepatocytes, thus causing damage to hepatocytes, but astragaloside IV plays a certain therapeutic role in reversing this damage.
研究领域:
药理学
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肝病学
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细胞生物学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
Frontiers in Oncology
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2021
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IF 6.244
Yang Jianru,Jia Yang,Wang Bing,Yang Shengrong,Du Kun,Luo Yujie,Li Yunhe,Zhu Bing
Background: The effects of up-regulated CircCHST15 on lung cancer remained unclear. In this study, the role of CircCHST15 in lung cancer was investigated.Methods: Dual-luciferase reporter verified the bioinformatics prediction that CircCHST15 targeted miR-155-5p and miR-194-5p. The correlation between CircCHST15 and PD-L1 was analyzed by Pearson analysis. CCK-8 and colony formation was performed to determine the viability and proliferation of lung cancer cells. After the lung cancer (subcutaneous-xenotransplant) model was established in mice, the T cell subtype and related cytokines in mouse tumor tissues were detected by flow cytometry and ELISA. Moreover, the expressions of CircCHST15, miR-155-5p, miR-194-5p, immune-related, and proliferation-related factors of the lung cancer cells or mice tumor tissues were detected by immunohistochemistry, RT-qPCR, or Western blot.Results: CircCHST15 and PD-L1 were high-expressed in lung cancer, and the two was positively correlated. CircCHST15 targeted miR-155-5p and miR-194-5p, the later further targeted PD-L1. Lung cancer cell viability and proliferation were increased by miR-155-5p and inhibited by miR-194-5p. CircCHST15 located in the cytoplasm promoted tumor growth, down-regulated the expressions of miR-155-5p and miR-194-5p, and up-regulated the expressions of PD-L1, Ki-67, PCNA, CCL17, CCL22, IFN-γ, TNF-β, and IL-10. Also, CircCHST15 decreased the CD8+ cells in mouse blood and tumor, but increased the Tregs in mouse tumor. PD-L1 inhibitor showed an opposite effect to CircCHST15 on mouse tumors.Conclusion: CircCHST15 sponged miR-155-5p and miR-194-5p to promote the PD-L1-mediated immune escape of lung cancer cells.
研究领域:
肿瘤学
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分子生物学
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免疫学
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产品关联
DOI
PubMed
高影响因子
FOOD AND CHEMICAL TOXICOLOGY
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2021
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IF 6.025
Yue Leng,Li Ren,Shu Niu,Tiehua Zhang,Jie Zhang
The endocrine disruptive capability of plasticizers to activate nuclear receptors has attracted great interest. This study is aimed to assess the potential glucocorticoid effects of metabolites of plasticizers. The effects of metabolites of plasticizers on the transcriptional activity of glucocorticoid receptor (GR) were investigated using reporter gene assays. All of them failed to exhibit agonistic/antagonistic effects on GR. However, a combination of dexamethasone and monobutyl phthalate (MBP) could synergistically activate GR. MBP combined with dexamethasone also enhanced GR nuclear translocation by Western blot , while mifepristone restrained GR cytoplasmic-to-nuclear translocation. MBP co-treated with dexamethasone resulted in synergistic induction of PEPCK and MKP-1 gene expression by real-time PCR and PEPCK protein level by Western blot. Furthermore, the carboxyl and ester groups of MBP have influences on the charge distribution of MBP, leading to change of electrostatic interactions between MBP and GR by calculations on electronic properties. Both hydrophobic and hydrogen bonding interactions play a crucial role in the stabilization between MBP and GR conducted by molecular docking and dynamics simulation. This work confirms that GR could remain stable upon binding to MBP. In conclusion, dexamethasone and MBP could synergistically activate GR, resulting in synergetic enhancement of subsequent GR-mediated endocrine disrupting effect.
研究领域:
毒理学
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内分泌学
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分子生物学
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药理学
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产品关联
DOI
PubMed
高影响因子
AQUACULTURE RESEARCH
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2023
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IF 2
Tingting Hou,Zaizhao Wang,Kui Tang,Shuai Zhang,Shilin Liu,Jialiang Liu,Xiaoteng Fan,Xiaolin Liu
This study investigated the capability for fin regeneration in zebrafish larvae with the mechanism of FCS isolated from green and purple sea cucumbers Apostichopus japonicus (G-FCS and P-FCS). HPGPC determined that mean molecular weight of purified G-FCS and P-FCS is 19.5 and 18.8 kDa, and the FT-IR spectrum displayed similar characteristic absorption peaks. AFM images presented that G-FCS was shown as a spherical polysaccharide, while P-FCS molecules exhibited elongated chains. For the regeneration examination, FCS increased the regrowth area of the larval fin at 48 hours postamputation with enhanced locomotor behaviors, and the transcription levels of regeneration-related genes (Wnt10a, msx1b, fgf20a, bmp2a, bmp4, and igf2b) were upregulated. Moreover, FCS promoted the mRNA expression of inflammatory factors (iNOS, TNF-α, IL-6, IL-10, and IFN-γ) and TLR/NF-κB pathway-related genes (TLR3, TLR4a, NF-κb, and MyD88). The Western blot analyses of TNF-α and IL-1β supported that FCS enhanced immune response. ELISA results indicated that FCSs increased the contents of NO, CAT, SOD, and G6PD. Summarily, the present results suggested that FCS was a potential regeneration enhancer by elevating immunomodulatory and antioxidative processes in zebrafish.
研究领域:
海洋生物学
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发育生物学
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免疫学
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生物化学
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产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）