Use of 4-phenylbutanoic acid derivatives in production of drugs for treating cerebral ischemia-reperfusion injury
A technology of cerebral ischemia-reperfusion and its derivatives, which can be applied in drug combinations, pharmaceutical formulations, cardiovascular system diseases, etc., and can solve the problems of secondary cerebral edema, intracranial pressure, unfavorable curative effect of non-monomers, and high price
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Embodiment 1
[0045] To test the cytotoxicity of compounds
[0046] The cytotoxicity of the compound was detected by the MTT method, and the specific steps were as follows: After the cells in good condition covered about 90% of the bottom area of the dish, the cells were subcultured into a 96-well plate, and 100 μL of cell suspension was added to each well to make each well into About 1x105 cells (cell counting method), 6 duplicate wells for each sample, and set up blank group and control group at the same time, add 100 μ L PBS buffer solution to each well of the edge well of the 96-well plate, and the plating is completed; wait until it is about 70% full Change the starvation medium every now and then to continue culturing for 22-24h;
[0047]After 24 hours of starvation, replace it with complete medium for culture, and administer drugs at the same time, and cultivate in a constant temperature incubator for 24 hours; after 24 hours, directly add MTT solution under dark conditions, 20 μL ...
Embodiment 2
[0050] Detection of compound viability on cells
[0051] Cell culture, passage, and plating were carried out according to the method in Example 1. TG (final concentration 10 μM) was given to each experimental well 1 h after pre-administration, and then placed in a constant temperature incubator for 24 h; after 24 h, MTT solution was directly added under dark conditions, 20 μL per hole, and the incubator was cultivated for 4 h; 4 h Afterwards, the original culture solution was discarded, 150 μL of DMSO was added to each well, and placed on a microwell shaker for 10 minutes to completely dissolve the blue crystals, and finally the OD value was measured at 490 nm in a microplate reader.
[0052] Cell survival rate / %=OD490 (administration group-blank group) / OD490 (control group-blank group)×100%
[0053] The result is as figure 2 As shown, the cell viability of each group was statistically significant for the TG group. It shows that these compounds have a certain reverse effec...
Embodiment 3
[0055] Detection of 4-PBA inhibitory activity by Western-blotting method
[0056] The expression of GRP78 and CHOP proteins in endoplasmic reticulum-stressed PC12 cells by the effect of compounds A-M by western blotting.
[0057] (1) Take out the densely growing cells in the logarithmic growth phase from the incubator, digest, subculture, and spread the cells into six-well plates.
[0058] (2) Before adding the stimulant, replace the overnight culture medium with fresh prepared medium, add 2 μL of different concentrations of drugs to each culture dish, and set up negative control DMSO and positive control TG. The final drug concentration was 10 μM.
[0059] (3) Protein samples were collected after drug incubation for 24 hours, 100 μL of lysis mixture was added, and lysed on ice for 20 minutes. Scrape the cells with a scraper, transfer them to an EP tube with a pipette gun, centrifuge at 12000r / min at 4°C for 5min. Take the supernatant and measure the absorbance at 595nm to ...
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