A composition for inhibiting macrophage activation and its application in preparation of anti-inflammatory products
A macrophage and composition technology, applied in the field of anti-inflammatory drugs, can solve problems such as cell apoptosis, and achieve the effects of delaying drug resistance, reducing concentration and reducing damage
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Embodiment 1
[0033] Different concentrations of cinnamide and inhibitor MDIVI-1 on Raw264.7 cells were measured by MTT experiments, as follows:
[0034] Raw264.7 cells were blown down from the culture bottle with a pipette, and cell suspension was made using 10% FBS DMEM medium to inoculate concentration 5 × 10 4 The concentration of / ml is inoculated on a 96-well plate (100 μL / well), placed in 5% CO 2 It was cultured for 24 hours in a 37 ° C incubator. The cinnamide and MdiVi-1 concentration were set to 80 μm, 40 μm, 20 μm, 10 μm, 5 μM, and four additional holes were disposed for each concentration. 8% CO 2 At 37 ° C for 24 h, after suction of the supernatant, 100 μl of concentration of 0.5 mg / ml of MTT solution (diluted with a 5 mg / ml MTT solution in the base DMEM was obtained); inserted into the incubator, continued to cultivate for 3 hours. Be careful to suck the supernatant in the hole. 100 μl of DMSO was added to each well, and the incubator was allowed to stand for ten minutes; t...
Embodiment 2
[0037] Lipopolysaccharide (LPS) is a component of the outer wall of Gram-negative bacterial cells, which has strong inflammation, but there is no direct toxic effect on macrophages. It has been widely used in a variety of inflammatory responses and oxidative stypes, including Raw264.7 cells in vitro model, so the following tests were tested using LPS modeling. Based on LPS (lipid polysaccharide) induction, cinnamarne (CMA) 20 μm, 10 μm and MDIVI-1 30 μm, 20 μm, and 10 μm performed separate and two-two combination drugs to produce NO content in Raw264.7. With the Griess experiment, the specific embodiments are as follows:
[0038] Raw264.7 cells were blown down from the culture flask using a pipette, and cell suspensions were made using 10% FBS DMEM medium to inoculate concentration 1 × 10 6 The concentration of / ml is inoculated on a 24-well plate (400 μL / well), placed in 5% CO 2 , Cultured in a 37 ° C incubator. After 24 hours, the medium, the blank group was added to 400 μl o...
Embodiment 3
[0042] Cinnamonal aldehydrates with Western blot experiments promote primary and autophagy, and specific embodiments are as follows:
[0043] 1. Cell treated before the protein: EtOAc. After 24 hours, the supernatant was discarded, and 2 ml of the fully medium containing cinnamarcal aldehyde (20 μm) was added. After 2 hours, LPS was added to give a final concentration of 1 μg / ml while setting the blank group and the LPS model group. After 24 hours of continuing, cell extraction can be performed.
[0044] 2. Cell total protein extract method: Raw264.7 cells in petri dishes were gently washed three times with PBS, transferred to 1.5 ml EP tube, centrifuged at low speed, and discarded PBS. The EP tube was placed on ice, and 100 μl of cell lysate was added, mixed with lysis of 10 minutes. The resulting supernatant is a cell total protein solution at a high speed and low temperature (4 ° C / 12000 rpm).
[0045] 3. Determination of protein concentration: Formulated protein standard s...
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