Application of alpha7 nicotinic choline receptor agonist in preparation of peptidyl arginine deiminase 4 inhibitor
A technology of peptide acyl arginine deiminase and choline receptor, which is applied in the field of α7 nicotinic choline receptor agonists in the field of preparing peptide acyl arginine deiminase 4 inhibitors, and can solve clinical problems. Efficacy remains to be verified, etc.
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Embodiment 1
[0021] Inhibitory effect of α7 nicotinic acetylcholine receptor agonist on the expression of PAD4 in macrophages
[0022] RAW264.7 cells were treated with 1×10 5 / mL density was seeded in 12-well plates, and the cells were placed at 37°C, 5% CO 2 After culturing in an incubator for 1 h, α7nAChR agonists PNU-282987, AR-R17779 (5, 10, 20 μM) and sinomenine (SIN, 0.25, 0.5, 1 mM) were added to the cell culture medium, respectively. After 30 min, phorbol ester (phorbol-12-myristate-13-acetate, PMA) or lipopolysaccharide (LPS, 1 μg / mL) was added to the cell culture medium. After 4 hours, the total protein of the cells was extracted, and the expression level of PAD4 was detected by Western blot. See the experimental results figure 1 , PNU-282987 (10, 20 μM), AR-R17779 (10, 20 μM), and sinomenine (0.5, 1 mM) significantly inhibited the expression of PAD4 in macrophages.
[0023] The experimental results showed that α7nAChR agonists significantly down-regulated the expression leve...
Embodiment 2
[0025] Inhibitory effect of α7nAChR agonists on the accumulation of intracellular catalytic products of PAD4
[0026] RAW264.7 cells were treated with 1×10 5 / mL density was seeded in 12-well plates, and the cells were placed at 37°C, 5% CO 2 After culturing in an incubator for 1 h, α7nAChR agonists PNU-282987, AR-R17779 (5, 10, 20 μM) and sinomenine (SIN, 0.25, 0.5, 1 mM) were added to the cell culture medium. After 30 min, PMA or LPS (1 μg / mL) was added to the medium. After 4 hours, the total cell protein was extracted, and the accumulation level of the intracellular catalytic product of PAD4 (citrullinated histone H3) was detected by Western blot. See the experimental results figure 2 , PNU-282987 (10, 20μM), AR-R17779 (10, 20μM), sinomenine (0.5, 1mM) significantly decreased the citrullination level of histone H3 in macrophages.
[0027] The test results showed that α7nAChR agonists significantly down-regulated the citrullination level of histone H3 in macrophages, an...
Embodiment 3
[0029] Inhibition of PAD4 activity by α7nAChR agonists
[0030] The α7nAChR agonists PNU-282987 (0.25, 0.5, 1, 2, 4, 8mM), AR-R17779 (0.25, 0.5, 1, 2, 4, 8mM), sinomenine (SIN, 1, 2, 4, 8, 16, 32mM) were added to the reaction buffer containing 100mM Tris (pH 7.2), 10mM calcium chloride, 10mM DL-dithiothreitol and 0.2ml recombinant PAD4 solution, and incubated at 37°C for 2min. A short peptide containing arginine and 10 mM N-α-benzoyl arginine ethyl ester were added to start the reaction, and then the reaction system was incubated at 37° C. for 30 min. The enzymatic reaction was terminated by adding 60% (w / v) perchloric acid. Ammonium ferrous sulfate hexahydrate and ammonium ferric sulfate dodecahydrate in 1N H 2 SO 4 The redox reagent prepared in was added to the reaction mixture, and then boiled for 10 min. After cooling to room temperature, the acid mixture (phosphoric acid, sulfuric acid and deionized water) and 12.5 mM 2,3-butanedione monoxime solution were added. Aft...
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