Application of n-acetyl-d-glucosamine in the preparation of drugs for preventing and treating Alzheimer's disease
A technology based on glucosamine and acetyl, which can be used in applications, drug combinations, antipyretics, etc. It can solve the problems that the effects of neurodegenerative diseases have not been reported, and achieve the improvement of cognitive memory impairment, cognitive function, and inhibition of inflammation The effect of the reaction
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Embodiment 1
[0031] Example 1 The effect of Ogt deficiency on the activation of astrocytes in vivo and in vitro.
[0032] 1. Method
[0033] Ogt floxp / floxp Mice with Glast-Cre ERT2 Mice were crossed to produce inducible Ogt conditional knockout (cKO) mice. Male mice were injected with tamoxifen 100 mg / kg or control agent (peritoneal injection, once a day, for 5 consecutive days) at 7 weeks for 8 weeks Afterwards, a series of experiments were carried out, with 3 rats in each group.
[0034]Isolation and culture of primary brain astrocytes from adult mice: Adult mice were dislocated cervically, quickly decapitated, dissected hippocampal and cortical tissues in pre-frozen PBS, and carefully peeled off the meninges under a stereomicroscope. After washing with cold PBS, the tissue was minced with a scalpel and then digested with 0.125% trypsin at 37°C for 15 min with rotation. Rotate slowly with 1000 U / mg papain for 15 minutes at 37°C, and then mix with 20 μl DNase I (50000 U / ml). The cel...
Embodiment 2
[0043] Example 2 Can the activation of astrocytes cause inflammatory response after Ogt deficiency.
[0044] 1. Method
[0045] Enzyme-linked immunosorbent assay (ELISA): hippocampal tissue homogenate was lysed in ice buffer (500μl, 20mM Tris-HCl, 150mM NaCl, pH7.4, 1% Triton X-100, 1mM EDTA, 1x protease inhibitor tablet) Centrifuge at 14,000 g for 15 min at 4°C, and collect the supernatant. Enzyme-linked immunosorbent assays of inflammatory factors IL-1β and TNF-α were performed with 100 μl of cell and tissue supernatants using an ELISA kit (Thermo Fisher Scientific).
[0046] Preparation of Ogt inhibitor (Ogt-I): Prepare Ogt inhibitor (5 mM) stock solution with DMSO, add Ogt inhibitor to the medium with a final concentration of 20 μM, and co-culture with purified adult astrocytes for 72 hours, An equal volume of PBS was added as a control.
[0047] 2. Results
[0048] To determine whether activated astrocytes release inflammatory factors, IL-1β immunostaining was perform...
Embodiment 3
[0051] Example 3 Effects of Activated Astrocytes on Hippocampal Neurons and Cognitive Function of Mice after Ogt Deficiency
[0052] 1. Method
[0053] Golgi staining of neurons and quantitative analysis of dendritic branching and spines: Golgi staining was performed according to the manufacturer's protocol (FD neurotechnologies, PK401). Briefly, fresh mouse brains were stored in Golgi-Cox solution in the dark for 14 days, and then incubated with 30% sucrose for 2 days at 4°C. Coronal sections were cut to 200 µm thickness using a cryostat. Sections were collected, dehydrated in absolute alcohol, washed in xylene, and covered with resin medium for mounting in sequence. Z-axis imaging of dendritic branches and dendritic spines of 6 to 8 pyramidal neurons in the CA1 region of each mouse was performed with an Olympus optical microscope. Only cells with cell bodies in the section that were clearly stained, had no broken dendrites, and did not overlap adjacent cells were selected...
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