Application of naringenin in preparation of accelerant for promoting polarization of M1 microglial cells to M2
A technology of microglia and naringenin, which is applied in the field of biomedicine to achieve the effects of removing toxic substances, inhibiting inflammatory reactions, and promoting transformation
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Embodiment 1
[0028] Example 1 Molecular docking study of naringenin and its target PPARγ
[0029] Method: The 3D structure of the compound naringenin was obtained from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ), and the core was screened from the RCSB PDB (http: / / www.rcsb.org / ) database For the crystal structure of the target protein, use pymol (version 2.4.0) to remove water molecules and small ligand molecules in the protein, and isolate the protein structure. The genetic algorithm in AutoDock 4.2.6 software was used for semi-flexible docking. The coordinates and size of the center of the box were set according to the position of the active site of the protein molecule and the area where it may act on the small molecule of the ligand. The rest of the parameters were kept at default. For the compound Molecular docking analysis with the target protein.
[0030] Results: Molecular docking is to simulate the interaction between the ligand small molecule and the receptor biomacromolecule, o...
Embodiment 2
[0031] Example 2 Naringenin promotes the polarization of M1 microglial cells to M2
[0032] Primary microglial cell culture: 5-6 ddY mice (P2-4), the cerebral cortex was separated after alcohol sterilization, the dura mater was removed, chopped and placed in DMEM (+FBS) medium, centrifuged (1000rpm, 3min), remove the supernatant, add 0.25% trypsin 2mL, at 37 degrees, 5% CO 2 Incubate in an incubator for 30min, add 4mL of 10% FBS-DMEM medium, centrifuge (2000rpm, 5min), remove the supernatant, add DNase I / Trypsine inhibitor 2mL, at 37 degrees, 10% CO 2 Incubate in an incubator for 15min, add 4mL of 10% FBS-DMEM medium, centrifuge (1000rpm, 3min), remove the supernatant, add 4mL of 10% FBS-DMEM medium, blow and dissociate the cells with a rubber dropper, Add 2 mL of 15% BSA in PBS solution from the bottom, centrifuge (2000 rpm, 6 min), remove the supernatant, add 2 mL of 10% FBS-DMEM medium, count the cells and inoculate them on a 6-cm culture dish (8*10 5 cells). After 14 da...
Embodiment 3
[0035] Example 3 Naringenin activates the phagocytosis of microglial cells on Aβ
[0036] Method: The experimental procedure is as follows image 3 As shown in A, the primary cultured microglial cells were seeded on 8-well plates (1.5*10 4 cells / well), add naringenin (0.1~100μM) or positive control IL4 after 1h, add Aβ1-42 (1μM) after 4 hours of culture and continue to culture for 24h, remove the supernatant containing Aβ and naringenin, add Aβ1 -42 Hilyte Fluor 488, cultured for 3 hours, fixed cells with 4% PFA, added primary antibody CD206 (1:200, M2 marker) or iNOS (1:50, M1 marker) and secondary antibody Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA) for immunofluorescence staining, photographed with a fluorescent microscope (BX-61 / DP70, Olympus) and analyzed with Metamorph software.
[0037] The result is as image 3 As shown in B, after the addition of Aβ1-42, the phagocytosis of fluorescently labeled Aβ1-42Hilyte Fluor488 by microglial cells was greatly increased, b...
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