Iranian Journal of Basic Medical Sciences
·
2017
·
IF 1.424
Jimei Bu,Hengbing Zu
Objective(s): In previous studies, researchers observed that doxepin could improve cognitive processes and has protective effects on the central nervous system. Thus, this study was designed to analyze the effects of doxepin on β-amyloid (Aβ)-induced memory impairment and neuronal toxicity in rat and to explore the underlying mechanism. Materials and Methods: Rats were treated with Aβ1-42 and doxepin was injected to validate its effects on cognitive function. The Morris water maze test was performed to detect memory function. Aβ1-42-treated SH-SY5Y human neuroblastoma cell line was also used to detect the effects of doxepin and to explore the underlying mechanism. Western blotting analysis was used to detect the protein expression levels of PSD-95, synapsin 1, p-AKT and p-mTOR in rats. Results: After treated with 1 mg/kg of doxepin, Aβ1-42-treated rats showed markedly lower escape latency and higher platform-finding strategy score. Low doses of doxepin significantly reversed the effects of Aβ1-42 on the protein expression levels of PSD-95, synapsin 1, p-AKT and p-mTOR in rats. In vitro experiment showed the consistent results. Besides, PI3K inhibitor (LY294002) treatment could markedly reversed the effects of doxepin on Aβ1-42-treated SH-SY5Y cells. Conclusion: Our results demonstrated that doxepin could protect against the Aβ1-42-induced memory impairment in rats. The protective effect of doxepin was associated with the enhancement of PSD-95 and synapsin 1 expression via PI3K/AKT/mTOR signaling pathway.
研究领域:
神经科学
·
药理学
·
认知科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Xue Zhao,Yunshan Wang,Jiatai Yin,Sidi Cun,Yuanyuan Ou,Qian Li,Xinfeng Zhao,Yajun Zhang
Background Protein immobilization method play a pivotal role in developing affinity chromatographic assays for both basic and applied research, and site-specific, covalent attachment serves as the most desirable strategy. Traditional strategy often gives rise to inconsistent results due to the use of heterobifunctional cross-linker or a similar two-step protocol. Introducing unnatural amino acids into the immobilization is possible to address the issue, however, it needs purification of the protein whereas suffers from loss of the protein activity. Herein, this work developed a method by integrating a thiol-ene addition click reaction during the protein expression for preparation of immobilized protein. Results The methodology involved the genetically incorporated O-allyl- l -tyrosine ( O -ALTyr) into serotonin transporter (SERT) and norepinephrine transporter (NET), the fusion proteins were immobilized onto silica gel through " thiol-ene " click reaction. The activities of the immobilized proteins were verified by surface plasmon resonance (SPR) measurement with high success rates of 91.4 % for immobilized SERT and 92.8 % for immobilized NET. The stability and repeatability of immobilized proteins were quantified by determination of protein-drug binding parameters by affinity chromatography under diverse conditions including extreme pHs (pH = 6.0–8.0) and high concentrations of denature reagents (5 %–20 % for DMSO and 5 %–40 % for methanol). Finally, screening analysis found that crocin I and n -butylphthalide were dual-target compounds binding to SERT and NET in Gardenia jasminoides Ellis - Rhizoma Ligustici Chuanxiong Hort (GR) extract. The accuracy of the chromatographic screening method was validated by determined the regulation of the two compounds on release of neurotransmitters including 5-HT, NE, and DA, as well as the expression of the SERT and NET. Significance This efficient, purification-free, site-specific, and one-step immobilization method proved to yield immobilized protein with highly enhanced stability and reproducibility which is possible to be used in challenging systems, especially affinity chromatographic analysis using mobile phase containing organic solvents. It benefits analysis of interaction between protein and drugs with low water-solubility, thereby having potential for strengthening the role of immobilized protein-based methods in many fields like drug discovery and fabricating other protein surface to pursue improved assay performance.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
eLife
·
2024
·
IF 6.4
Lu Yang,Zijin Lin,Ruijing Mu,Wenhan Wu,Hao Zhi,Xiaodong Liu,Hanyu Yang,Li Liu
Blood–brain barrier (BBB) prevents neurotoxins from entering central nervous system. We aimed to establish and characterize an in vitro triple co-culture BBB model consisting of brain endothelial cells hCMEC/D3, astrocytoma U251 cells, and neuroblastoma SH-SY5Y cells. Co-culture of SH-SY5Y and U251 cells markedly enhanced claudin-5 and VE-cadherin expression in hCMEC/D3 cells, accompanied by increased transendothelial electrical resistance and decreased permeability. Conditioned medium (CM) from SH-SY5Y cells (S-CM), U251 cells (U-CM), and co-culture of SH-SY5Y and U251 cells (US-CM) also promoted claudin-5 and VE-cadherin expression. Glial cell line-derived neurotrophic factor (GDNF) levels in S-CM and US-CM were significantly higher than CMs from hCMEC/D3 and U-CM. Both GDNF and US-CM upregulated claudin-5 and VE-cadherin expression, which were attenuated by anti-GDNF antibody and GDNF signaling inhibitors. GDNF increased claudin-5 expression via the PI3K/AKT/FOXO1 and MAPK/ERK pathways. Meanwhile, GDNF promoted VE-cadherin expression by activating PI3K/AKT/ETS1 and MAPK/ERK/ETS1 signaling. The roles of GDNF in BBB integrity were validated using brain-specific Gdnf silencing mice. The developed triple co-culture BBB model was successfully applied to predict BBB permeability. In conclusion, neurons enhance BBB integrity by upregulating claudin-5 and VE-cadherin expression through GDNF secretion and established triple co-culture BBB model may be used to predict drugs’ BBB permeability.
研究领域:
神经科学
·
细胞生物学
·
药理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2026
·
IF 7.3
Yunshan Wang,Nan Lu,Peiyao Wu,Yue Chu,Yonghao Xing,Jing Wang,Xinfeng Zhao
The development of conventional orthosteric drugs targeting G protein-coupled receptors (GPCRs) is often hindered by off-target effects and low subtype selectivity. Allosteric ligands enable precise control of receptor signaling through selective conformational changes, highlighting their critical importance as a promising therapeutic strategy and as a key frontier in modern drug discovery. To enhance GPCR ligand screening efficacy, surface-enhanced Raman spectroscopy (SERS), with sensitivity and narrow spectral peaks, was employed to outline a methodology based on allosteric aptamer binding and stabilizing conformations of the β2 adrenergic receptor (β2AR) for identifying GPCR ligands by fabricating a sandwich-type SERS sensor. The method involved immobilization of β2AR on magnetic plasmonic nanoparticles by label-free click chemistry, probing and stabilization of the allosteric conformational changes by RNA aptamer, and amplification and detection of the signal by SERS-active nanoparticles. The success of the allosteric β2AR assay was confirmed through multiple analytical techniques, including SERS, SEM, UV–vis spectroscopy, and particle size analysis. This methodology demonstrates broad applicability, characterized by rapid response, high sensitivity, and excellent selectivity. BI167107 could be detected within a range of 10–9–10–6 M, with a detection limit of 49 pM. Such a sandwich biosensor successfully identified the agonist, antagonist, and allosteric inhibitor of β2AR from the ligand complex in both aqueous solution and serum. This method is expected to become an alternative for high-throughput and multiplexed drug screening when it comes to a compound library.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）