Application of disulfiram in preparing drugs for preventing and treating diseases related to NLRP3 inflammasome
A technology of inflammasome and disulfiram, which is applied in the field of medicine, can solve problems such as deficiency and achieve strong pharmacological effects
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Embodiment 1
[0055] Example 1 In vitro study of the inhibitory effect of disulfiram on NLRP3 inflammasome
[0056] 1. On the first day, divide J774A.1 cells into 96-well plate, each well 5×10 5 cells;
[0057] 2. Seed the plate overnight, discard the supernatant, add 100 μL of DMEM medium containing 10% serum containing bacterial lipopolysaccharide (LPS) (1 μg / ml) to each well, and then add different concentrations (0 μM, 2 μM, 5 μM, 10 μM) DSF was treated for 1 hour, and then nigericin (Nigericin, 10 μM) or ATP (5 mM) was added for 1 hour;
[0058] 3. After the treatment in step 2, collect the cell supernatant, and use the Moμse IL-1βELISA kit to measure the IL-1β content. The operation steps are carried out according to the kit instructions. The results are shown in figure 1 .
[0059] figure 1 The results showed that under the joint action of LPS pretreatment and the second signal Nigericin or ATP, the inflammasome was activated, and IL-1β was matured and secreted into the supernata...
Embodiment 2
[0060] Example 2 DSF inhibits the activation of NLRP3 inflammasome
[0061] 1. On the first day, divide the J774A.1 cells into a 6-well plate, each well 2×10 6 cells;
[0062] 2. Seed the plate overnight, discard the supernatant, add 1 mL of DMEM medium containing 10% serum containing bacterial lipopolysaccharide (LPS) (1 μg / ml) to each well, then discard the supernatant, and add different concentrations of (0 μM, 2 μM, 5 μM, 10 μM) DSF opti-MEM medium for 1 hour, then add nigericin (Nigericin, 10 μM) for 1 hour;
[0063] 3. After the treatment in step 2, collect the cell supernatant (Supernant) and cell lysate (Lysate), extract protein from the supernatant and lysate according to conventional methods, use anti-IL-1β antibody, anti-Caspase-1 antibody, anti-NLRP3 antibody and anti-NLRP3 antibody ASC antibody for western blot analysis, see the results figure 2 .
[0064] figure 2 The results showed that under the joint action of LPS pretreatment and the second signal Nige...
Embodiment 3
[0065] Example 3 In vivo study on the inhibitory effect of disulfiram on NLRP3 inflammasome - DSF inhibits LPS-induced peritonitis
[0066] 1. Take 6-8 week-old C57BL / 6 female mice, weigh them, and number them. Then intragastric administration of DSF, 100 mg / kg per mouse, and intragastric administration of PBS containing 10% DMSO in the control group, once a day, for three consecutive days, after intragastric administration of DSF for 1 hour on the third day, intraperitoneal injection of LPS, 20 mg / kg per mouse;
[0067] After 2 and 6 hours, the eyeballs were removed for bloodletting, and the blood was collected. After the bloodletting, the mice were killed by cervical dislocation. Let the blood stand at room temperature for 30 minutes, then centrifuge at 3000rpm for 30 minutes to collect serum;
[0068] 3. Rinse the abdominal cavity with 1ml of normal saline, and centrifuge the suctioned normal saline at 3000rpm for 5 minutes to collect the supernatant;
[0069] 4. The seru...
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