Antidiabetic medicament screening method using Nur77-LKB1 interaction as target point
A nur77-lkb1, anti-diabetic technology, applied in biochemical equipment and methods, testing pharmaceutical preparations, biological testing, etc., can solve the problem of low phosphorylation activity of AMPKα in the liver, and achieve intuitive effect, stable system, and drug screening mechanism clear effect
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Embodiment 1
[0058] Compounds that inhibit the Nur77-LKB1 interaction were screened by co-immunoprecipitation and Western blotting.
[0059] The test method is as follows:
[0060] 1) Cell transfection and drug treatment:
[0061] The cells were inoculated on a 6-em-diameter culture dish, transfected 24 hours later, and the cells were replaced with fresh culture medium before transfection. The transfection steps are: take a 1.5mL centrifuge tube, add 180μL of water, the corresponding plasmid, 2.5M CaCl 2 20 μL, HBS 200 μL, mix gently, let stand at room temperature for 15-30 minutes, then slowly add dropwise to the culture medium, shake the culture plate gently to make the distribution even.
[0062] After 36 hours of transfection, the cell culture medium was replaced with serum-free DMEM (Dulbecco's Modified Eagle's Medium), and the corresponding drugs were added. In the control group, the medium was changed at the same time, and an equal volume of drug solvent dimethyl sulfoxide (DMSO)...
Embodiment 2
[0087] In the further screening method, Western blotting was used to detect the effect of the compound to be screened in Example 1 on the phosphorylation level of AMPKα in normal mouse liver cells AML12.
[0088] The test method is as follows:
[0089] 1) Cell Lysis
[0090] Cells were seeded on a culture plate, and after 24 hours of culture, treated with drugs for an appropriate time, discarded the culture medium, washed once with PBS buffer, and then added PMSF and Cocktail containing 1mM (add just before use, add 1μL for every 100μL lysate) lysisbuffer cell lysate (50mM HEPES (pH 7.4), 100mM NaCl, 10% glycerol, 1% Triton-X-100, 1.5mM MgCl 2 , 25mM NaF) 400μL, sonicate on ice, centrifuge at 13000rpm at 4°C for 30min, collect the supernatant, measure the protein concentration and perform western blotting (method refer to Example 1).
[0091] 2) Determination of protein concentration
[0092] a. Add 200 μL Bradford protein assay solution to each well of the 96-well plate, a...
Embodiment 3
[0101] The method of measuring blood glucose by blood sampling from tail vein of mice was used to determine the hypoglycemic ability of TAPA1 on type II diabetes mouse model db / db mice.
[0102] The test method is as follows:
[0103] Take 16 hyperglycemic db / db mice (male, 8 weeks old) and randomly divide them into the experimental group (TAPA1 administration group; first dissolve TAPA1 in DMSO to make 1M mother solution, then take the required amount and 3 times the volume The emulsifier Tween-80 is mixed evenly, after standing for 15min, slowly add normal saline, flick to make the TAPA1-DMSO-Tween-80 mixture slowly dissolve in the normal saline to form an aqueous solution) and the control group (solvent group: use DMSO Replace TAPA1-DMSO solution, the configuration method is the same as above). Administered by intraperitoneal injection, the dose of TAPA1 was 50mg / kg, the mice in the solvent group were injected with the same volume of solvent as the experimental group, once...
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